ITS2 marker is highly efficient in species discrimination but its application in DNA barcoding is limited due to huge variations in the PCR success rate. We have hypothesized that higher GC content and the resultant secondary structures formed during annealing might hinder the PCR amplification of ITS2. To test this hypothesis, we selected 12 species from 12 different families in which ITS2 was not amplified under standard PCR reaction conditions. In these samples, DMSO, formamide, betaine, and 7-deaza-dGTP were evaluated for their ability to improve the PCR success rate. The highest PCR success rate (91.6%) was observed with 5% DMSO, followed by 1 M betaine (75%), 50 μM 7-deaza-dGTP (33.3%), and 3% formamide (16.6%). The one sample that did not amplify with DMSO was amplified by adding 1 M betaine. However, combining DMSO and betaine in the same reaction did not improve the PCR. Therefore, to achieve the highest PCR success rate for ITS2, it is recommended to include 5% DMSO by default and substitute it with 1 M betaine only in the case of failed reactions. When this strategy was tested in 50 species from 43 genera and 29 families, the PCR success rate of ITS2 increased from 42% to 100%.
The genus Melia L., which belongs to the 'Mahogany' family Meliaceae, is a source of important phytochemicals with marked medicinal properties. Species identification in Melia is complex due to the existence of overlapping morphological features. Though Melia dubia Cav. is listed as a synonym of Melia azedarach L., it is not clear from the available literature whether they are the same species or different, and the species complexity still remains unresolved. In the present study, ten accessions of M. dubia and M. azedarach were analysed by DNA barcoding using three chloroplast DNA markers (rbcL, matK, and trnH-psbA), and one nuclear marker (ITS2). Intra-specific divergence was not found in any of the four markers. However, the inter-specific divergence between M. azedarach and M. dubia ranged between 0.3% (rbcL) and 4.7% (ITS2) for individual markers, and for the combined dataset, it was 8.5%. Among the four markers, ITS2 was found to be the most suitable marker for
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