DNA barcoding, a technique projected for rapid identification of unknown biological samples which uses short (known as ‘Folmer’ region which has 658 bp long, present at the 5′ end of the CO1 mitochondrial genome) and agreed upon DNA sequences [25–26]. Upon its first initial success in more than 200 allied Lepidopteran species, it has been considered as a powerful tool for identification of all eukaryotes at the species level. However, it was found that CO1 gene was not suitable for plants because as such there is no region of genome, cytoplasm or nuclear that could be identified. The plant mitochondrial genes with low nucleotide substitutions and low evolutionary rates were considered unsuitable for barcodes of plants [27–30]. Therefore, the present study was initiated to check the applicability of nuclear genome (ITS) for identification, authentication of some Vandaceous orchids based on the earlier recommendation made by Plant Working Group of consortium for the Barcode of Life (CBOL) and Barcode of Life Database (BOLD) standard guidelines [30]. The sampled specimens (individuals) collected from different parts of Nagaland during the field survey were brought to the Department laboratory and stored at -20°C in a deep freezer to minimize the degradation of DNA and to preserve them till DNA was extracted. For DNA isolation, CTAB method [18] protocol was followed for some species. However, some orchid species accumulate mucilage (Acampe, Aerides, Arachnis) to conserve water and as food reserve [31]. The presence of high mucilage (polysaccharides and polyphenols) contents in such species was the major obstacle in DNA isolation and PCR amplification. Therefore, a modified CTAB method [19] in concentration and a step-wise manner was modified for those species which has high mucilage content.
The amplification success rate for ITS was 91.25% in the present study, a relatively higher as compared to other workers [28, 32–33]. A higher amplification rate of 97% was reported by Roy et al. [34] in the tested samples of 11 species of Ficus and 4 species of Gossypium and also Singh et al. [35] while testing the congeneric species of Dendrobium with 98.97% amplification success rates was reported.
Following amplification, the successful amplicons are packed, labeled and send it for sequencing after completing all the formalities/company instruction to various laboratories to sequence the desire size/targeted loci. The sequence success rate and the total number of barcode sequence generated was 91.78% and 67 respectively.
Intra and Inter-Specific Variations
For correct identification and generation of DNA barcodes for species, the assessment of intra and inter-specific variations is important [23–24, 36]. The minimum inter-specific variation is greater than the maximum intra-specific variation and the difference between the two is referred as ‘barcode gap’ [37]. The intra and inter-specific divergence were evaluated/expressed in terms of K2P distances as done by Chen et al. [38] and also Parveen [33]. The intra-specific variations were evaluated for 25 species that were represented by more than one individual. Variable range of intra-specific distances was obtained in all different species. The minimum/lowest divergence among the investigated species was observed in Cleisostoma williamsonii while the maximum/highest divergence was observed in Acampe ochracea. While evaluating the inter-specific divergence for 7 genus that were represented by more than one species in a genus, it was observed that the genus Arachnis (2 species) with an average inter-specific K2P Distance of 0.032 (Ranges from 0-0.063) was the lowest while Acampe with 0.187 (ranges from 0-0.431) was the highest.
Species Discrimination Rates and Evaluation of DNA Barcodes
For evaluating species resolution and selecting DNA barcodes three methods were used viz., genetic distance, phylogenetic tree method and BLAST analysis. The genetic distance employs the assessment of intra and inter-specific divergence. The intra and inter-specific divergence should not overlap in an ideal barcode. The difference/gap between the two specific divergences provides a perfect barcode which is referred as barcode gap [25–26, 36]. The phylogenetic tree method is constructed using sequences from the targeted locus and the percent species resolution was determined by cluster analysis [36, 39]. The species for which all the individuals clustered together in a single clade are considered as unequivocally identified species and those which clustered with the individuals of the other species were treated as unresolved. During the present analysis, the species resolution of the investigated loci calculated using both these methods showed different results.
The last method used for evaluating species discrimination is the BLAST analysis [40]. In this method, the unknown individual barcode sequence is search in the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for a very similar/identical sequence from the database, containing reference barcodes of correctly identified species.
In spite of low amplification rate, ITS showed more species discrimination rates of 90.90% and 95.45% by distance based method and phylogenetic tree method. At the genus level, ITS BLAST hits increases from 79.40-95.52%. The overall species discrimination rates are 89.15%. The high species discrimination ability of this region could be due to its high rate of evolution leading to genetic changes that allows differentiation of closely related congeneric species [28, 35, 41–42]. No out-group was use in the phylogenetic tree construction so as to find/compare the genetic closeness of this closely related species, which are similar in vegetative characters and difficult to identify based on morphological characters.