Feasibility of nuclear ribosomal region ITS1 over ITS2 in barcoding taxonomically challenging genera of subtribe Cassiinae (Fabaceae)

Abstract

Premise of the StudyThe internal transcribed spacer (ITS) region is situated between 18S and 26S in a polycistronic rRNA precursor transcript. It had been proved to be the most commonly sequenced region across plant species to resolve phylogenetic relationships ranging from shallow to deep taxonomic levels. Despite several taxonomical revisions in Cassiinae, a stable phylogeny remains elusive at the molecular level, particularly concerning the delineation of species in the genera Cassia, Senna and Chamaecrista. This study addresses the comparative potential of ITS datasets (ITS1, ITS2 and concatenated) in resolving the underlying morphological disparity in the highly complex genera, to assess their discriminatory power as potential barcode candidates in Cassiinae.MethodologyA combination of experimental data and an in-silico approach based on threshold genetic distances, sequence similarity based and hierarchical tree-based methods was performed to decipher the discriminating power of ITS datasets on 18 different species of Cassiinae complex. Lab-generated sequences were compared against those available in the GenBank using BLAST and were aligned through MUSCLE 3.8.31 and analysed in PAUP 4.0 and BEAST1.8 using parsimony ratchet, maximum likelihood and Bayesian inference (BI) methods of gene and species tree reconciliation with bootstrapping. DNA barcoding gap was realized based on the Kimura two-parameter distance model (K2P) in TaxonDNA and MEGA.Principal FindingsBased on the K2P distance, significant divergences between the inter- and intra-specific genetic distances were observed, while the presence of a DNA barcoding gap was obvious. The ITS1 region efficiently identified 81.63% and 90% of species using TaxonDNA and BI methods, respectively. The PWG-distance method based on simple pairwise matching indicated the significance of ITS1 whereby highest number of variable (210) and informative sites (206) were obtained. The BI tree-based methods outperformed the similarity-based methods producing well-resolved phylogenetic trees with many nodes well supported by bootstrap analyses.ConclusionThe reticulated phylogenetic hypothesis using the ITS1 region mainly supported the relationship between the species of Cassiinae established by traditional morphological methods. The ITS1 region showed a higher discrimination power and desirable characteristics as compared to ITS2 and ITS1 + 2, thereby concluding to be the locus of choice. Considering the complexity of the group and the underlying biological ambiguities, the results presented here are encouraging for developing DNA barcoding as a useful tool for resolving taxonomical challenges in corroboration with morphological framework.

Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM) Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs

“…The primer pairs of ITS1 investigated in studying the Cassinae group generally resulted in the successful amplification of amplicons from remarkably more species than those of ITS2 . Following the results of large-scale meta-analysis ITS1 claimed to be better DNA barcode than ITS2 ( Mishra et al, 2016b ). Moreover, due to lower rate of nucleotide substitution in the chloroplast genome as compared to the nuclear genome, it provides less variable sites for analysis.…”

“…Based on a previous study ( Mishra et al, 2016b ) and the results from the present dataset, ITS1 barcode reflected significant sequence divergence among the seven Senna species and were thus selected for subsequent HRM analysis. Conserved regions flanking the variable sites were identified visually and HRM-suited potential primers were designed with AlleleID (v.7.7, Premier Biosoft International, Palo Alto, CA, United States) using its SYBR Green Design function.…”

“…Seven species with three individuals ( n = 3) were used for study. Total genomic DNA from the field samples of reference species was isolated following the protocol described by Mishra et al (2016b) . The genomic DNA from commercial samples (HSA01–HSA10) was isolated with DNeasy ® PlantMini Kit (Qiagen, Valencia, CA, United States) using the protocols supplied with the kit.…”

Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia

“…The species-specific monophyly demonstrated through tree-based analysis adds to its usefulness as a locus of choice in Terminalia . The greater discriminatory power at low taxonomic levels and higher evolutionary rate of nuclear region ITS makes it a promising locus in plant molecular systematics [ 58 ]. In addition, matK is one of the most rapidly evolving coding sections of plant genome with higher discrimination power than rbcL [ 59 ].…”

Employing barcoding markers to authenticate selected endangered medicinal plants traded in Indian markets

“…The utility of ITS/ITS2 was amply demonstrated and a strong case for its inclusion in the core barcode comprising matK and rbcL was advocated by China Plant BOL Group (2011). Rather in one of the recent studies, ITS1 exhibited higher species discrimination power than ITS2 among the members of taxonomically complex sub-tribe Cassiinae of the family fabaceae (Mishra et al 2016). In our previous study, all the 36 investigated species of Dendrobium could be discriminated on the basis of ITS alone (Singh et al 2012).…”