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 is one of the most valuable groups all over the world, identification of animals [8, 9]. However and is also an impressively large and complex family of flowering . 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]

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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 rate of amplicons of the ITS [5, 16] may such reasons as the presence of intra- ITS with another sequence showed be due to these characteristics. The ITS genomic variability, the divergent a greater ability to identify species, regions were evaluated and found to have paralogous copies (multiple functional the ITS sequence alone was still an high-quality bidirectional sequences [5]. copies) within individuals and effective barcode among proposed loci

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[rbcL, matK, atpF-atpH, rpoB, rpoC1, get better results than individual ones ability of ITS2 for differentiating trnH-psbA, trnL-F, and ITS] [25]. [41-43]. With a higher taxonomic medicinal Dendrobium species from The ITS was employed successfully level (section, genus, subgenus, tribe, one another and also from non- in Dendrobium (Orchidaceae). subfamily), ITS, matK, trnL and rbcL orchids and adulterants. In 2010, the Specifically, the phylogenetic were most often used [44-49], e.g. three 50790 ITS2 sequences of plants were relationship and the differentiation of subgenera of Cymbidium genus could downloaded from GenBank and were 11 medicinal Dendrobium spp. from one be distinguished clearly from among ten evaluated according to their sequence another and from two adulterant species species by its ITS (ITS1+5.8S+ITS2) lengths, GC content, intra- and inter- Pholidotaarticulata and Flickingeria [50]. specific divergence, and efficiency comate could be analysed using this locus of identification. The study proposed In general, the ITS is worth its use [20]. The specific nucleotide sequences of that the ITS2 locus shows significant for barcoding projects; and in order to the ITS is used for the identification and sequence variability at the species increase resolution effects, ITS2 can be phylogeny of 20 Dendrobium species, level or lower, and should be used as a in which ITS1 and ITS2 regions exhibit an alternative solution, or supplementary universal DNA barcode for identifying more variation than the 5.8S rDNA markers maybe accompanied as plant species. Among them all, the [19]. The single ITS barcode revealed discussed in the combination barcodes success rates for using the ITS2 region to be the best DNA barcode affording latter. to identify Orchidaceae taxa at the 100% species resolution based on 129 ITS2: Located between ribosomal species level (%) were variable in congeneric species of Dendrobium, and genes 5.8S and 28S of the ITS regions, different genera Scaphyglottis 100.0, 93% based on sets of sequences from recently ITS2 has been highly noticed Satyrium 98.3, Dendrobium 91.9, both the experiment and the GenBank. as a valuable barcode for many plants. Dichaea 81.8, Disa 79.7, Masdevallia This resolution value of the single ITS The ITS2 spacer provides structural 79.6, Paphiopedilum 76.6, Telipogon barcode was higher than other single elements necessary for correct pre-rRNA 76.1, Cymbidium 74.1, Dendrochilum barcodes matK, rbcL, rpoB, rpoC1 and processing and probably has a function 71.2, Cyrtochilum 69.3, Phalaenopsis even combined barcodes [matK+rbcL] in the regulation of the transcription 65.9, Oncidium 65.1, Maxillaria 62.9, and [matK+rpoB+rpoC1] (33, 20, 18, of active ribosomal subunits [51]. As Gomesa 49.1, Diuris 31.1, and Ophrys 17, 80.77 and 92.31% respectively) in an identifying marker, this sort of ITS 22.7 [54]. In the study on 43 samples of the study [16]. Using nuclear ribosomal fragment does not only receive benefits Dendrobium, the ITS2 analyses showed a ITS sequence data, genetic units in from the long ITS possess but also significant divergence between the inter- Grammatophyllum speciosum complex overcomes the limits of a full-length and intra-specific genetic distances, and (Orchidaceae) were totally recognised at region. the presence of a barcoding gap was species level to be G. speciosum Blume, obvious [51]. The variability of ITS2 G. wallisii Rchb.f., G. kinabaluense In ITS2, the success rates of both was sufficient enough to distinguish Ames and C. Schweinf., G. pantherinum PCR and sequencing were very high even closely related species. The Rchb.f., and G. cominsii Rolfe [26]. [3], mostly 100% in Dendrobium [51] or phylogenetic analysis of ITS2 regions 93.8% in a wide range of plants instead of of 64 Dendrobium species also showed In orchid phylogenetic research, 42.3% for full ITS [52]. This is because good results when the cluster analysis there have been many different markers the length of ITS2 is quite short at about mainly supported a relationship between used, among them is ITS, which has 248 bp in Dendrobium [51], and the the species of Dendrobium established usually been a favourite choice of design of the universal primers for this by traditional morphological methods researchers [27-31]. The ITS tree of 16 sequence is easy due to the availability and many previous molecular analyses Paphiopedilum species and two varieties of flanked conserved regions (5.8S and [51] (Table 2). found in Vietnam, received strong 26S rRNA). This characteristic was trnH-psbA: trnH-psbA is a non- Jack-knife support for phylogenetics also ideal for barcoding, which should coding intergenic spacer which is a analysis [32]. However as discussed be short enough to recover amplicons in the barcoding field, not any single rapidly evolving region. trnH-psbA from degraded DNA [23, 53] and can or multiple-locus markers could was early on highly evaluated by overcome the trouble of the universal totally identify the species, and so the Kress, et al. (2005) [5] because of its ITS from contaminated microorganism combination of regions in phylogenetic high interspecific variation, high length [53]. studies could not get the strong support variation, good priming sites, and it was in all clades of phylogenetic trees [33- The rate of successful identification proposed to be the most viable candidate 35]. The ITS was used and combined with the ITS2 is high at 92.7% at the for a single-locus barcode for land plants with other regions in most of the species level and 99.8% at the genus identification [55]. Actually, trnH-psbA phylogenetic studies thus far [3, 36- level [52]. Early from 2001, Lau, et al. was quite easily amplified with a success 40]. The combined data matrices often (2001) [53] discovered the significant rate that either might reach 100% in

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large numbers of land plants [5, 56] or barcoding gap was obvious, and so the not only significantly complex, but also at least more than 90% in a wide range locus was evaluated as an effective spacer significantly different in length, and of plants [3, 7, 52, 56-58]. In orchids for barcoding Dendrobium species and those were the reasons for the difficulty particularly, PCR rates were also very for differentiating Dendrobium species of alignment for analysis. This problem high, up to 100% in Dendrobium [59, from other adulterating species. But in either led to the failure of sequence 60], Oncidium [61], Holcoglossum [14], 2011, the species resolution of trnH- alignment or the requirement of manual and Cymbidium [62] (Fig. 2). psbA on Dendrobium was 79.3% over editing [7]. 504 samples (lower than matK 88.8%, trnH-psbA was popularly known The length of trnH-psbA in a wide atpF-atpH 82.4%, and rbcL 79.8% [63], to have a high sequence divergence range of plants is quite short at about 400 or 8.14%, which is lower than the ITS, [5, 55, 56] due to its large number of bp, to satisfy the criteria of the barcode, ITS2, and matK [60]. On Holcoglossum insertions and deletions (indels). The which should be short enough for easy (Orchidaceae), 52 individuals belong species resolution rates of this region in amplification [5, 7]. On the other hand, 12 species were analysed for barcode plants were the highest in many research there was research that showed that with six markers rbcL, matK, atpF- the short length of trnH-psbA makes studies, for examples (82.6%) out of the atpH, psbK-psbI, trnH-psbA and ITS. a lack of information for barcode and nine other loci ITS, rbcL, ndhJ, matK, Species resolution of trnH-psbA was phylogenetic analysis [56]. In orchids, rpoB2, rpoC1, ycf5,accD [55] and (59%) 5/12 species (using Neighbor-Joining the length of trnH-psbA are 850 bp in out of the nine other loci cox1, 23S algorithm), equal to ITS 5/12 and lower Dendrobium (included entire regions rDNA, rpoB, rpoC1, rbcL, matK, atpF- than matK 6/12 [14]. atpH, psbK-psbI] [58]. In the big DNA psbA-trnH 722-785 bp plus regions of barcoding project of the CBOL Plant In general, trnH-psbA was highly rpl12 - 279 bp and rps19 - 19 bp) [59], Working Group, trnH-psbA also showed evaluated early-on, but less favourable 739 bp in Holcoglossum [14]. However, the highest species discrimination (69%) at a later time due to some obvious the containing of rpl12 and rps19 in in comparison with another six loci problems. The most common complaint orchids and some monocots caused (atpF-atpH, matK, rbcL, rpoB, rpoC1, was about trnH-psbA. That it was much longer in size of this region up psbK-psbI) from 259 samples of 95 generally too difficult to align in land to > 1000 bp and meets the problem of species of 34 genera seed plants [7]. plants [5, 55] as well as in Orchidaceae. PCR and sequencing [7, 65]. The high Species resolution rate of trnH-psbA in trnH-psbA possesses many indels difference in length caused multiple other land plant researches was 67.6%, in their sequences [5, 55, 56]. The bands in few samples of Dendrobium second high among five screening mononucleotide (A/T) repeats (or known that hard to recognise which one is markers psbA-trnH, matK, rbcL, rpoC1, as homopolymers) and/or small tandem correct trnH-psbA segment, and the ycf5, ITS2, ITS [52]. repeats (AT) were frequently noted in bad quality sequence made this locus this intergenic spacer [3, 7, 55, 56]. The excluded from the analysis [16]. In Orchidaceae, species resolution existence of homopolymers (AAAA/ In another point of view, the difficulty of this sequence varied from different TTTTTT) for most non-coding regions, of alignment was not a major obstacle genera and different studies. Among including trnH-psbA, interrupted the [5] compared to the benefit provided by six markers (trnH-psbA, accD, rpoC1, sequence runs and caused problems their sequence information. Indels are rpoB, matK and ndhJ), only trnH-psbA with obtaining high-quality bidirectional useful pieces of information for species could resolve 8 of 11 Mesoamerican sequences [3, 7, 58, 64], especially with discrimination, i.e. they could help orchid species (72.7%), which was just the forward primer, as in Dendrobium to distinguish three species of genus lower than matK (10/11). Proportion of [16]. The results were both lower and Solidago, which cannot be separated due monophyletic species recovered with overlapped between bidirectional reads to low sequence divergence, and Kress, UPGMA of trnH-psbA were 90.6%, or only allowed partial sequences to et al. (2005) hoped for an improvement the same as matK, and the two highest be obtained. This is also another major of DNA barcoding tools for which to among eight loci accD, ndhJ, matK, limitation for this locus. In orchids and utilize the indel information [5]. Thus, trnH-psbA, rbcL, rpoB, rpoC1, and some other monocots, beside of these this locus is still valuable for many ycf5 for 172 individuals of 86 species similar problems of indels and repeats research projects, to be used as potential (including 71 individuals of 48 orchid [16], the genomic rearrangement of the barcodes, especially with a combination species + other angiosperm species) non-homologous inverted repeat has of supplement barcodes, which will be [57]. On Dendrobium, in testing the been also found [15, 55], and especially discussed later. unique marker trnH-psbA, Yao, et al. the insertion of well-conserved exon- (2009) calculated the intergenic spacers copies of rpl12 and rps19 (known as matK: matK is the gene coding for of all species (0.3 to 2.3%) and the pseudogene) [57, 59, 65]. The indels, the maturase K protein. This is also a intraspecific variation (0 to 0.1%) [59]. repeats and inverted repeats likely in rapidly evolving gene [7] that potential This research’s result meant that the trnH-psbA sequences made this region as an identification molecular marker

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that can be used in many barcoding very high range 95.23% ([6] - Indian ycf1b (85%), which was better than and phylogenetic studies. This region Paphiopedilum), 92.31% ([14] - any of the matK, rbcL, and trnH-psbA was evaluated at much higher levels Holcoglossum), and 99.32% ([16] - and even slightly better than the core of sequence variations for species Dendrobium). Then, matK is still a good barcode matK+rbcL (71.31%) [15]. The discrimination [7, 65]. Lahaye, et al. choice for the barcoding of orchids, reason that this valuable region received (2008b) [66] suggested that matK was especially if good primers could be little attention for DNA barcoding or the preferred universal barcode for developed. molecular systematic purposes at low flowering plants, including orchids and taxonomic that is ycf1 is too long (5709 rbcL: rbcL is the ribulose-1,5- re-affirmed best potential of this region bp in Nicotiana tabacum) and is too bisphosphate carboxylase/oxygenase in Lahaye. The results of DNA barcode variable to permit the design of universal large subunit gene in the plastid genome. library for 20 endangered Orchidaceae primers. However, the high variability of The single region of rbcL was not high species distributed in Mexico using ycf1 indicates its potential value in DNA favoured because it is too long (1428 the barcodes matK and rbcL showed barcoding of land plants. Phylogenetic bp [5]) and contains highly conserved that single matK allowed for the application of ycf1 had been found for regions [61]. As with high universality, identification of the most orchid species Orchidaceae [30, 49, 71, 72] and was the amplification rates of rbcL were [67]. The species resolution of matK evaluated as the most variable and rather good in most of the studies, from was 100% for Paphiopedilum [6], 5/5 parsimony-informative among five other 90 to 100% in a broad range of plants [3, species for five medicinal Dendrobium chloroplast genes (matK, rbcL, rpoC1, 5, 15, 55, 57, 63, 64], and also in orchids species [68] and 6/12 for Holcoglossum rpoC2 and ycf2) [71, 73]. ycf1 should be [6, 14, 16, 60]. Although rbcL could get [14] in comparing with other studied more tested in molecular identification high-quality bidirectional sequences single regions. of orchids. and well universality [7, 63], this poor matK has also been proved to have variable region could discriminate well atpF-atpH: Recently, Kim, et al. the same problem as trnH-psbA with at the genus level, but could not show (2015) [74] suggested the intergenic homopolymer runs of mononucleotide adequate resolution at the species level spacer atpF-atpH as a barcoding repeats for some taxonomic groups and in plants [3, 5, 64, 70] and in orchids marker after concluding a 100% species led to low-quality bidirectional sequences particularly [57]. In contrast with PCR discrimination ability of this region on [3, 58, 68]. However this rate was not success rates, resolution rates were very 28 individuals of four species of Korean significant, and only a few samples of low [69.8% - [55], 75% - [64], 79.8% - Cypripedilum-Orchidaceae. The results matK amplification in Dendrobium gave [63], 58.02% - [15], 26.4% - [3] and not obtained from such a set of observations multiple bands [16]. But matK suffered suitable for molecular identification in were found, such as sequence variations, most by a low amplification success many Orchidaceae species (Oncidiinae, species-specific SNPs, indels differences, rate. From 96 individuals, at 96 species Paphiopedilum, Holcoglossum and length variations, and the use of species- of 48 genera of land plants, the PCR Dendrobium) [6, 14-16, 25, 61]. specific primers (ARMS method for rate of matK was just 39.3%, far lower However, rbcL was highly considered amplification refractory mutation than other screening loci (trnH-psbA, in many combined barcode, as discuss system). The authors also suggested rbcL, ITS1, ndhJ, rpoB2, rpoC1, ycf5, later. identification using electrophoresis accD) [55]. Fazekas, et al. (2008) used based on length variations of sequences. ycf1: Another locus that is “more 10 primer pairs for sequencing reactions atpF-atpH has significant length variable than matK” [71] or “any existing of matK, but the success rate could still variations among species and was plastid candidate barcodes and can serve not covered all the samples (88%) [58]. used for molecular identification and as a barcode for land plants”, that was The poor PCR recovery might due to the phylogenetic study of low taxonomic ycf1, proposed as a barcode by Dong, nonuniversal primers. level plant species, although it does et al. (2015) [15]. Within the plastid not function independently [15, 21]. This problem could be overcome genome, ycf1 spans the small single However, this region was not suitable for through design improvements or by copy (SSC) and the inverted repeat (IR) Holcoglossum orchid DNA barcoding improving the specific primers [7, 65, regions. The section of ycf1 in the IR study [25] and fell for recovery of high- 68]. Using the specific primer pairs region is short (less than one kilobase quality bidirectional sequences [7]. 390F and 1326R from Cuénoud, et al. long) and conserved. In contrast, the [69], it could get 100% amplification of section of ycf1 in the SSC region has rps16-trnQ: Jhong-Yi Lin and his matK [57]. But particularly in orchids, high sequence variability in seed plants group have explored that the rps16-trnQ the amplification rates of matK are quite [15]. ycf1 was known to be absent from marker showed the best discrimination good. These rate could be up to 100% some genera, but exists in orchids, power and was considered to be the ([61] - Oncidiinae, [62] - Thailand including ycf1a and ycf1b. 357 of 420 best DNA barcode in the study. Another Cymbidium, [60] - Dendrobium) or at tree species could be distinguished using 15 of 27 studied cpDNA markers were

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also recognised as highly variable good discriminatory power but had for a wide range of plants as well as among moth orchids, with polymorphic the lowest sequencing success in orchids, it was clear that no single locus information contents of 8.0 and were these trials, and substantial problems could be sufficient in this role for both suggested to combine with rps16-trnQ generating bidirectional reads [7]. The universality and resolvability, and multi- [75]. Orchidaceae, psbK-psbI showed the locus barcodes seem to be more robust highest mean to be interspecific at a K2P trnL-F: Orchidaceae, trnL-F regions and effective. As Kress, et al. (2005) has distance (0.1192), followed by matK were evaluated to be effective rabbet DNA suggested, it may need more than one (0.0803), atpF-atpH IGS (0.0648), trnH- barcode marker gene [25]. trnL-F was locus for species-level discrimination psbA IGS (0.0460) and rbcL (0.0248) also used in many phylogenetic studies of [5]. [44]. By overcoming the obstacles and orchid taxa such as Orphrys, Angraecinae, difficulties of the intergenic spacer as In 2007, the two-locus barcode , Arethuseae, Vandae, discussed in trnH-psbA, this region can trnH-psbA+rbcL was first proposed Bulbophyllum, Coryciinae, Cypripedium, be used as a potential molecular marker by Kress and Erickson [55] with the Tangtsinia, and Orchiade [2, 29, 40-42, for orchids. species resolution increased to 85% for 76-81]. angiosperms comparing to the highest Multi-locus barcodes psbK-psbI: Like trnH-psbA, the 82.6% of single locus trnH-psbA. intergenic spacer psbK-psbI showed In an effort to find universal barcodes Actually, the combination of trnH- psbA+rpoB or trnH-psbA+rpoC1 was 85% too, but the PCR success rate of rbcL in the study was higher than rpoB and rpoC1, and then the totally result of trnH-psbA+rbcL was better. In the same year 2007, Chase, et al. first suggested the three-locus barcodes either of matK+rpoC1+rpoB or matK+rpoC1+trnH-psbA [65]. The combination of two or three loci of matK, rpoC1, rpoB and trnH-psbA was also recommended for 11 Mesoamerican orchid species in this year and the resolution results showed that matK+rpoC1+rpoB 100% (discriminate 11/11 species), matK+rpoC1+trnH- psbA and matK+rpoB+trnH-psbA 90.9% (10/11 species), and the single matK 90.9% (10/11 species). In the study of Singh, et al. (2012) on Dendrobium (Orchidaceae), the combination matK+rpoC1+rpoB gave the highest resolution (94.44%) among other three- locus barcodes, just lower than the ITS (100%). From this result, they suggested that “barcodes, if based on the single or limited locus, would be specific taxa” [16]. On the subject Cymbidium (Orchidaceae), both the two options 1) The matK+rpoC1+trnH-psbA, and 2) The matK+rpoB+trnH-psbA have Fig. 2. Gene map of Cymbidium chloroplast genome [82] with notations for potential Fig. 2. Gene map of Cymbidium chloroplast genome [82] with notations for succeeded 100% species resolution of 19 loci fopotentialr barcod lociing for(Wit barcodingh Inverte (Withd re pInvertedeat regi repeatons (I Rregionsa and (IIRab );and sm IRalb);l s ismallngle copCymbidiumy in Thailand [62]. However, (SSC)single and lcopyarge (SSC)singl eand cop largey (L SCsingle) re gicopyons . (LGSC)enes regions. on the Genes outsid one theof t houtsidee map arwithoute rpoC1 or rpoB, the two-locus transofcr ithebed map clockwi are transcribedse and g eclockwisenes on theand genesinside on of the t hinsidee map of theare map tra narescri bematKd +trnH-psbA barcode achieved counttranscribederclockwis ecounterclockwise.. Loci in rectang Lleoci sh inap rectanglees are poten shapestia l arema potentialrkers fo cusedmarkers in thonlyis moderate improvement (90.9%) reviewfocused). in this review). in comparing with the single matK Multi-locus barcodes In an e ort to nd universal barcodes for a wide range of plants as well as orchdis, JUNE 2017 • Vol.59 Number 2 Vietnam Journal of Science, 67 it was clear that no single locus could be sucient in this role for both universality and Technology and Engineering resolvability, and multi-locus barcodes seem to be more robust and e ective.As Kress, et al. has suggested, it may need more than one locus fosr pecies-level discrimination [5]. In 2007, the two-locus barcode trnH-psbA+rbcL was rst proposed by Kress and Erickson [55] with the species resolution increased to 85% for angiosperms comparing to 11 life sciences | biotechnology

resolution (90.6% of 48 orchid species (2009) tested on three loci rbcL, matK and the core barcode rbcL+matK gave plus 38 angiosperm species) [57]. trnH-psbA whether the use of multilocus the lowest resolution (49.7%) among supermatrices to generate phylogenetic In 2008, Fazekas, et al. selected a other two-locus barcodes, the highest hypotheses at the species-level would more loci combinations for barcoding Amplification discrimination rateMolecular of the markersthree-locus Study Studied regions Samplesimprove the resolution power.Sequence The separation (%) 92 species of land plants. A multilocus (% success) barcode was 81.8%recommendation by matK+trnH- results showed that the core-combining plant barcoding region should have psbA+ITS. rbcL+matK has high species barcode proposed by CBOL rbcL+matK multiple regions chosen from among identi fication power at the species level just discriminated 92%, while trnH- three of the coding (rbcL , rpoB, matK) in just some taxonomic groups (e.g. psbA+rbcL did 95%. The three-locus and two of the non-coding regions Orchidaceae ). The project proposed that combination rbcL +matK+trnH-psbA (trnH-psbA, atpF-atpH) (61-69%). As the ITS or ITS2 should be incorporated discriminated 98% [64]. rbcL+matK all combinations assessed using four into the core barcode (rbcL , matK) for was 93.1% species resolution and to seven regions had only marginally seed plants [3]. The combination of rbcL+matK+trnH-psbA was 95.3% [63]. different success rates (69-71%); values matK +ITS showed a greater ability Fazekas, et al. (2012) also suggested that were approached by several two- to identify species than matK or the the combination of rbcL+matK as the and three-region combinations (61- ITS alone in Holcoglossum [14], in core barcode with another supplement 69%) [58]. This meant that no single Dendrobium and Paphiopedilum [60]. barcode (ITS or trnH-psbA) [83]. combination clearly outperformed all On the subject Oncidium genus, a others. This situation was also proved in In 2010, ITS2 and psbA-trnH combination of trnH-psbA +trnF-ndhJ the study of Hollingsworth, et al. (2009) sequences were highly evaluated, with was proposed as a potential barcode by [21] with some three-locus combination 93.8% and 23.8% PCR success rates correct phylogenetic placement of 13/15 of rbcL, rpoC1, matK, and trnH-psbA. in 1,433 species of 551 genera in 135 Amplification Oncidiinae hybridMolecular varieties markers [61]. As Study Studied regions Samplesfamilies from four phyla (Angiosperms,Sequence separation (%) In 2009, the Consortium for the (% success) recommendation Gymnosperms, Ferns and Mosses), intergenic spacers were recognized to Barcode of Life (CBOL) Plant Working respectively, while its ITS fragments be high variable, Kim, et al. (2014) [44] Group first recommended the two-locus were only successfully amplified in also suggested the combination of three combination of rbcL+matK as a plant 42.3% of the experiments; and the intergenic spacers atpF-atpH+psbK- barcode. This combination represented identification rate of the psbA-trnH psbI +trnH-psbA as the best option for a practical solution to a complex trade- region was 96.5% at the genus level barcoding of the Korean orchid species, off between universality, sequence using the nearest distance method; the resolution up to 98.8%, among 26 quality, discrimination, and cost [7]. 72.8%, at the species level. ITS2+psbA- possible combinations of the five regions Now it is generally agreed that a plant trnH was strongly recommended as a rbcL , matK, atpF-atpH, psbK-psbI and barcode will combine more than one core and complementary barcode for a trnH-psbA . locus (5-7), including a phylogenetically broad series of plant taxa [52]. conservative coding locus (rbcL) with In general, the use of combined barcode could give better resolution one or more rapidly evolving regions On the contrast with CBOL (2009) (partial matK gene and the intergenic [7], in the BOL project in 2011 on in most but not all cases depending on spacer trnH-psbA). Thus Kress, et al. 1,757 species of seed plants in China, taxon specification (Table 2).

Table 2. Summary of studies comparing DNA barcoding regions in plants.

Amplification Molecular markers Study Studied regions Samples Sequence separation (%) (% success) recommendation

ITS, trnH-psbA, trnH-psbA, atpB-rbcL, psbM- Sequence divergence: Set 1: 19 rpl136-rpf8, 100% trnD, trnC-ycf6, - ITS (2.81%) species/8 trnL-F trnL-F, - trnH-psbA (1.24%) genera/7 trnC-ycf6, trnH-psbA, ITS trnk-rps16, - rpl136-rpf8, trnL-F (0.44%) families of ycf6-psbM 90% trnV-atpE, - atpB-rbcL (0.63%) angiosperm Other regions = 73- Kress, rpl36-rps8, - trnC-ycf6 (0.55%) 80% et al. ycf6-psbM (2005) [5] Set 2: 83 individuals/83 trnH-psbA = 100%, ITS, rbcL, species/72 rbcL = 95%, trnH-psbA>>rbcL trnH-psbA genera/50 ITS ≤ 88% families of angiosperm

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Taberlet, more than 100 trnL 67.3% et al. trnL, P6 loop plant species P6 loop 19.5% (2006) [84]

trnH-psbA (82.6%) trnH-psbA, rbcL, trnH-psbA = 95.8% 96 individuals/ ITS (81.5%) ITS1, ndhJ, matK, rbcL = 92.7% 96 species/48 rbcL (69.8%) rpoB2, rpoC1, ycf5, rpoC1 = 83.3% Kress and genera/43 Other loci (≤ 70%) accD of accD, rpoB ≈ 80% Two-locus barcode: trnH- Erickson families of land trnH-psbA+rbcL, angiosperms, ndhJ = 70%, psbA+rbcL (2007) [55] plants rnH-psbA+rpoB2, (85%) gymnosperms, ferns, ITS1 = 60.4% rnH-psbA+rpoC1 mosses, and ycf5 = 50% Other pairs of two loci liverworts matK = 39.3% (≤ 82.5%)

Chase, Three-locus barcode: et al. rpoC1+rpoB+matKor (2007) [65] rpoC1+matK+trnH-psbA 172 individuals/86 trnH-psbA, (90.6%) Lahaye, species (48 matK accD, ndhJ, matK, All other regions = et al. orchid species matK+trnH-psbA (90.9%) matKor rbcL, trnH-psbA, 95-100% (2008a) +38 species Other loci (≤ 87.5%) matK+trnH-psbA rpoB, rpoC1, ycf5 (except ycf5 and ndhJ) [57] from 13 All barcodes combine angiosperm (93.1%) families) accD, ndhJ, matK, Lahaye, rbcL, trnH-psbA, 101 et al. rpoB, rpoC1, ycf5, individuals/38 matK (2008b) atpF-atpH, psbK- species [66] psbI

% sequencing success trnH-psbA (59%) 23S rDNA, rbcL = matK (56%) 100% (2 primer pairs atpF-atpH, psbK-psbI (45%) used) rbcL, rpoB (42-48%) trnH-psbA = 99% cox1, matK, 251 cox1 (10%) rpoC1 = 95% (3 Combinations of 3-4 loci Fazekas, 23S rDNA, rpoB, individuals/92 23S rDNA (7%) primer pairs used) from: rbcL, rpoB, matK, et al. rpoC1, rbcL, trnH- species/32 rpoB = 92% (5 primer trnH- psbA, atpF-atpH (2008) [58] psbA, atpF-atpH, genera of land rpoB+rpoC1 (50%) pairs used) psbK-psbI plants matK+atpF-atpH+psbK-psbI matK = 88% (10 (69%) primer pairs used) rbcL+trnH-psbA, (64%) psbK-psbI = 85% matK+atpF-atpH cox1 = 72% rpoB+rpoC1+matK (61%) atpF-atpH = 65%

rpoC1 (38%), rpoB (40%), atpF-atpH (50%), matK (57%), rbcL (58%), atpF-atpH, matK, 907 samples trnH-psbA (58%) CBOL rbcL, rpoB, rpoC1, from 550 psbK-psbI = 77% psbK-psbI (64%) rbcL+matK (2009) [7] psbK-psbI, trnH- species genera all others = 90-98% 2-locus combinations psbA seed plants (59-75%) 3-locus combinations (65-76%) All 7 loci combination (73%) rbcL+matK (72%) atpF-atpH, matK, Hollingsworth, some combination of rbcL, rpoB, rpoC1, et al. (2009) rbcL, rpoC1, matK, trnH- psbK-psbI, trnH- [21] psbA. psbA

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matK (99%) trnH-psbA (95%) rbcLa (75%) matK+rbcL (92%) trnH-psbA+rbcL (95%) 1,035 rbcLa = 93% Kress, rbcL+matK+trnH-psbA (98%) rbcL, matK, trnH- samples/296 trnH-psbA = 94%, but et al. When both sequences rbcL+matK+trnH-psbA psbA species/181 problems sequencing (2009) [64] recovery and correct genera of plants matK = 69% assignment were taken into account: Of the 286 species trnH-psbA (90%) rbcLa (70%) matK (69%) Dicotyledons (76.1%) 50,790 plant and (74.2%) Yao, et al. 12,221 animal gymnosperms (67.1%) ITS2 ITS2 (2010) [54] ITS2 sequences ferns (88.1%) GenBank mosses (77.4%) animals (91.7%) 5,905 species/1,010 At specie level: genera/219 ITS2 (92.7%) families/7 phyla psbA-trnH, matK, psbA-trnH = 92.8% psbA-trnH (67.6%) Chen, et al. (Angiosperms, ITS2 or rbcL, rpoC1, ycf5, ITS2 = 93.8% (2010) [52] Gymnosperms, ITS2+psbA-trnH ITS2, ITS ITS = 42.3% At genus level: Ferns, Mosses, ITS2 (99.8%) Liver-worts, psbA-trnH (> 95%) Algae and Fungi) ITS (67.2%) ITS2 (54.6%) rbcL (26.4%) trnH-psbA+ITS (79.1%) trnH-psbA+ITS2 (69.7%) matK+ITS (75.3%) 1,757 matK+ITS2 (66.1%) species/141 rbcL = 94.5%, rbcL+ITS (69.9%) BOL rbcL, matK, trnH- genera/75 matK = 91.0%, rbcL+ITS2 (58.5%) ITS/ITS2 supplement to (2011) [3] psbA, ITS families/42 trnH-psbA = 90.2%, rbcL+matK (49.7%) core barodes rbcL, matK orders seed ITS = 88.0% matK+trnH-psbA+ITS (81.8%) plants matK+trnH-psbA+ITS2 (75.0%) rbcL+matK+ITS (77.4%) rbcL+matK+ITS2 (68.5%) rbcL+matK+trnH-psbA (62.0%) rbcL+matK+trnH-psbA+ITS 82.8% rbcL+matK+trnH-psbA + ITS2 (77.2%) matK (88.8%) 2,130 atpF-atpH (82.4%) Burgess, rbcL+matK, rpoC1, sequences/436 rbcL (79.8%) rbcL = 91.4% et al. trnH-psbA, atpF- species/269 trnH-psbA (79.3%) rbcL+matK rpoC1 = 74.5% (2011) [63] atpH genera of land rpoC1 (73.1%) plants rbcL+matK (93.1%) rbcL+matK+trnH-psbA (95.3%) Combination 5 loci (97.3%) Core [matK+rbcL] + Fazekas, supplements (ITS, trnH- et al. psbA) (2012) [83]

ycf1-a, trnK, rpl32-trnL, trnH-psbA, followed by Dong, et al. 23 loci present in at trnSUGA-trnGUCC, (2012) [85] least three genera petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, 3

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rpoB-trnC, psbE-petL, and rbcL-accD at genus level Set 1: 91species/5 orders dry medicinal product and At the species level: herbarium ITS (89.2%) specimens ITS2 (79.2) Han, et al. ITS2 = 91% ITS , ITS2 Set 2: 12861 ITS2 (2013) [23] ITS = 23% ITS and ITS2 At the genus level: sequences/ ITS (97.5%) 8,313 species/ ITS2 (93.8%) 8,313 species from 1699 genera, GenBank ycf1b (73.97%) 1352 sequences rbcLb (58.02%) of matK, rbcL rbcLb = 99.18%, matK (57.56%) and ycf1 from matK = 91.43%, rbcLb+matK (71.31%) 420 species/179 ycf1 (ycf1a, cf1b), ycf1b = 85.31% ycf1b+ rbcLb (81.39% ) Dong, et al. genera/76 plant rbcL, matK , trnH- ycf1b+matK (79.83%) ycf1 (ycf1a, ycf1b) (2015) [15] families. psbA ycf1b+rbcLb+matK (86.33%)

7 relatively well-sampled ycf1b highest plant groups

accD 3/11 (27.3%) matK 10/11 (90.9%) ndhJ 1/11 (9.1%) rpoB 6/11 (54.5%) trnH-psbA, rpoC1 4/11 (36.4%) Gigot, Combination 2 or 3 of 11Mesoamerican accD, rpoC1, trnH-psbA 8/11 (72.7%) et al. All = 100% rpoC1, rpoB, matK, trnH- orchid species rpoB, matK, rpoC1+rpoB+matK 11/11 (2007) [86] psbA ndhJ (100%) rpoC1+matK+trnH-psbA 10/11 (90.9%) rpoB+matK+trnH-psbA 10/11 (90.9%)

Intergenic variation of all 17 Dendrobium Yao, et al. species 0.3 to 2.3% psbA-trnH species, 1 psbA-trnH = 100% psbA-trnH (2009) [59] Intraspecific variation 0 to adulterance 0.1% trnH-psbA,matK, Wu, et al. trnF-ndhJ, ycf1- 15 Oncidiinae Correct phylogenetic All = 100% trnH-psbA+trnF-ndhJ (2010) [61] trnR, accD, rbcL, hybrid varieties placement of 13/15 varieties rpoB, rpoC1 ITS, matK (100%) Parveen, 8 species + 3 rbcL, 100% ITS, matK, rbcL, ITS (50%) et al. hybrids rpoB, matK rpoB, rpoC1 rbcL (25%) (2012) [6] Paphiopedilum rpoC1 rpoB, rpoC1 (12.5%) matK = 95.23% rbcL lowest rbcL = 100% matK 6/12 rbcL, matK, atpF- matK = 92.31% ITS 5/12 Xiang, atpH, psbK-psbI, ITS = 100% trnH-psbA 5/12 matK or et al. 12 species trnH-psbA, ITS of trnH-psbA = 100% matK+ITS 7/12 matK+ITS/ITS2 (2011) [14] Holcoglossum atpF-atpH (low) matK+trnH-psbA 6/12 psbK-psbI (low) ITS+trnH-psbA 6/12 matK+ITS+trnH-psbA 7/12

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11 Dendrobium, Wu, et al. ITS 2 adulterant ITS = 100% 100% ITS (2012) [20] species Chiang, 20 Dendrobium et al. ITS ITS = 100% ITS species (2012) [19] ITS (100%) matK (80.56%) rpoC1 = 100% Set 1: 292 rpoB (55.56%) matK = 99.32% individuals/36 rbcL (41.67%) rpoB = 99.2% species rpoC1 (38.89%) ITS = 98.97% Dendrobium matK+rpoB+rpoC1 (94.44%) rbcL = 96.91% Singh, matK, rbcL, rpoB, matK+rbcL (86.11 %) et al. rpoC1, trnH-psbA, ITS, matK+rpoB+rpoC1 (2012) [16] ITS ITS (100%) matK (76.92%) Set 2: 52 species rpoB (51.2%) (36 studied rpoC1 (42.31%) species + rbcL (38.46%) Genbank) matK+rpoB+rpoC1 (92.31 %) matK+rbcL (80.77%)

Siripiyasin, 19 species matK, rpoB, rpoC1, trnH-psbA+matK+rpoC1 et al. (2012) Cymbidium All = 100% All 100% sepcies resolution trnH-psbA trnH-psbA+matK+rpoB [62] Thailand Yukawa, 4 genetic units in Discriminate 4 different et al. Grammatophyllumsp ITS All = 100% ITS species of Grammatophyllum (2013) [26] eciosum complex trnH-psbA (83.5%) rbcL, matK, atpF- rbcL (60.5%) Kim, et al. 89 species of atpF-atpH+psbK- atpH, psbK-psbI and All = 100% atpF-atpH+ psbK-psbI+trnH- (2014) [44] Orchidaceae psbI+trnH-psbA trnH-psbA psbA (98.8%)

Set 1: 64 species 85.9% (by BLAST1 method), Feng, et al. ITS2 from ITS2 = 100% 82.8% (by nearest genetic ITS2 (2015) [51] Dendrobium distance method) trnN-rpl32, petN- 2 species trnN-rpl32, petN-psbM, psbM, petA-psbJ, Phalaenopsisap petA-psbJ, trnF-ndhJ, trnF-ndhJ, trnE- hrodite subsp. All = 100% trnE-trnT, accD-psaI, trnT, accD-psaI, Formosanaand rps15-ycf1, psbA-trnK, rps15-ycf1, psbA- P. amabilis atpF trnK, atpF Lin, et al. petN-psbM 16/19 (2015) [75] petA-psbJ 16/19 petN-psbM, petA- trnT-psbD 16/19 psbJ, trnT-psbD, 19 moth orchids trnF-ndhJ 16/19 trnF-ndhJ, trnN- All = 100% rps16-trnQ species trnN-rpl32 16/19 rpl32, rps16-trnQ, rps16-trnQ 19/19 rps16 rps16 15/19 trnL 18/19 Kim, et al. rpoC2, 4 species of rpoC2 = 100% 100% atpF-atpH (2015) [74] atpF-atpH Cypripedium atpF-atpH = 100%

ITS (31.93%) ITS2 (22.29%) matK (10.48%) Set 1: 184 Xu, et al. ITS, ITS2, matK, trnH-psbA (8.14%) species All = 100% ITS+matK (2015) [60] rbcL, trnH-psbA rbcL (5.56%) Dendrobium ITS+matK (76.92%) ITS+matK+trnH-psbA (73.13%) ITS2+matK (64.84%) matK+rbcL (24%)

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Some measurements for evaluating further tested [56, 74]. Bioinformatics references to screen for a new locus, effects of molecular markers tools for barcoding should be developed from plastid genome, to mitochondrial to use indel information [5]. genome and to nucleus genome. Different metrics to evaluate the molecular markers are usually discussed Some studies are also concerned The combinations of multi-locus in reference studies. It often suggests about the GC content of the sequences barcodes are now highly considered that the sequence lengths should be short [20, 54]. GC content is found to be as one of the improvement solutions enough (400-800 bp) for DNA extraction, variable with different organisms. DNA to obtain the best resolution results. amplification, and sequencing, but with high GC-content is more stable Many factors should be cared about certainly must be long enough to contain than DNA with low GC-content (Mega such as how many and which ones sufficient information for sequence net/help). Sequence length variation is would be combined. The final aim is to divergence [14, 57]. The sequence also a helpful feature in some cases [54, both maximise the loci to get the best should possess conserved flanking sites 74, 75]. Kim, et al. (2015) even suggests efficiency, and minimise the loci to for developing universal PCR primers using electrophoresis to identify species decrease cost and time (e.g. selection of a [55] but routinely retrievable with a based on length variation [74]. PCR- 2-locus barcode is based on costs and can single primer pair [7]. Easy alignment is based (multiplex and ARMS) method prevent further delays in implementing also one of the considered criteria [57] which determine specific SNPs has been a standard barcode for land plants) [7]. although in the situation of trnH-psbA used in analyses of sequence The selections of combining loci depend and some other intergenic spacers which [19, 74]. The secondary structure of on the characteristics of each locus. No are known as so variable that hard to the ITS2 region could provide useful single locus has shown high levels of align, the difficulty of alignment is not information for species identification universality and resolvability [21], and a major obstacle when comparing with and could be considered as a molecular no single barcoding region has an ability their advantages of variation sites [5]. morphological characteristic [54]. to resolve species to the same degree as nearly any of the multilocus barcoding The most concerned factor to To achieve optimal effect for methods [58]. The combination may identifying loci is good discriminatory barcoding, the utilization of many include a phylogenetically conservative power [3, 14, 16, 62]. This power is different pieces information is coding locus (rbcL) with one or more either based on sequence divergence necessarily, such as barcode setting rapidly evolving regions (part of the or variability [5, 55, 57]. The potential gap, length variation, indel variation... matK gene and the intergenic spacer parsimony-informative characters or DNA barcodes can be very effective in trnH-psbA). Chloroplast genome known as nucleotide substitutions are the context of a clearly circumscribed sequences contain regions that are the ones that much contribute to the floristic sample or plant community, highly variable, and this variability divergence between sequences [56, 74, and that additional data, such as of chloroplast genes differs markedly 83]. The one with the most features geography and morphology may be among genera [75, 85]. However, used to measure significant species- required to obtain higher rates of species primers designing for these intergenic level genetic variability and divergence identification in other contexts [64]. regions are the challenges with barcodes is “DNA barcoding gap”, which is Conclusions which, if based on the single or limited presented between intra- and inter- locus, would be specific taxa. So Singh, specific variations. High interspecific, Since the last classification of et al. (2012) [16] have recommended but low intraspecific divergence, are Orchidaceae in 2003, there has been the use of whole chloroplast genome expected to achieve maximal species major progress in the determination as single locus barcode in future discrimination sequencing [6, 14, 16, of relationships, despite that almost will help. Improve the effective of 20, 26, 51, 54, 57, 59, 60, 62, 86]. Indel all of the problematic placements available potential markers which low fragments (insertions and deletions) recognised in the previous classification amplification rate with specific primers also contain much useful information 11 years ago have now been resolved was one of the choices, e.g. matK much for identification work [20], e.g. it can by molecular methods [9]. However higher levels of sequence variation help to distinguish three species of barcoding for the identification of plants, and so possess high ability of spices genus Solidago despite low sequence as well as orchid species, still faces discrimination, but need improve PCR divergence [5]. Nucleotide substitutions many problems and needs improvement. primers [57]; in some cases, single- or account for about 70% and indels account These improvements are now continued multiple-primer sets are necessary [3, for about 30% of all mutations in the to achieve by different ways. New DNA 87]. chloroplast genome [56]. However, this regions more potential and suitable information is still not used effectively that can overcome the available limits To accurately determine the for available bioinformatics tools. The are ongoing investigated. Complete relationship between either the relative amounts of indels need to be sequenced DNA genomes are used as species or higher taxonomic level,

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