Identification of plant species is essential for their usage, and conservation of plant biodiversity. In the case of closely related species, the conventional method of identification using taxonomic evidence is challenging. Therefore, for rapid and accurate identification of plant species, the usage of molecular-based techniques is becoming increasingly evident (de Boer et al., 2022; Hebert et al., 2003). DNA barcoding is one such molecular technique used to identify and classify organisms based on the analysis of a short specific DNA region. This approach has revolutionized the field of taxonomy, as it provides a quick and reliable method for species identification. Plant chloroplast DNA markers, such as matK, rbcL, and trnH-psbA, have been widely used as DNA barcodes for plants (Kress, 2017). In addition, recently many researchers have shown that the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA is highly effective and reliable for the differentiation of plants at species level which shows the highest discriminating power (97% and 74% at the genus and species level respectively)(Yao et al., 2010).
In 2011 China Plant BOL group did a comparative analysis of a large dataset from diverse plant species and recommended an internal transcribed spacer (ITS) (Figure I) as a core barcode for seed plants (D. Z. Li et al., 2011;X. Li et al., 2015). The internal transcribed spacer is as a spacer region situated between the genes encoding the smaller and larger subunit of rRNA. Specifically, ITS1 is positioned between the 18s rRNA and 5.8s rRNA genes, consisting of an average length of 600 base pairs. On the other hand, ITS2 is located between the 5.8s rRNA and 26s rRNA genes, with an average length of 400 base pairs (Han et al., 2013; Tripathi et al., 2013). In the past decade, between ITS1 and ITS2, the ITS2 region of the internal transcribed spacer has been widely used DNA barcode for plant species identification due to its shorter length and and the ease of sequencing (Wang et al., 2018). The ITS2 region provides several advantages over the whole ITS or ITS1 region. It exhibits greater interspecific variation, has a shorter length, and provides a higher success rate in both PCR amplification and sequencing (Gu et al., 2013)(Han et al., 2013). Additionally, another drawback associated with using the complete ITS or ITS1 is the risk of fungal contamination during PCR amplification. Due to the substantial similarity between the ITS regions of fungi and plants, there is an increased chance of amplifying fungal ITS.). Therefore, in the recent decade, ITS2 has been accepted as the ideal DNA barcode by the plant scientist community and largely used to identify and classify a wide range of plants from weeds to medicinal plants (Khan et al., 2019; Zhao et al., 2018; Zuo et al., 2011).
Fig. I Schematic representation of ITS region of the nuclear ribosomal RNA (nrRNA) genes.
gDNA – genomic DNA, ETS- External Transcribed spacer, ITS1 and 2 – Internal Transcribed spacer, ITS2F and ITS2R – Forwrard and Reverse primers to amplify ITS2 region
For an ideal DNA barcode, the availability of a universal primer is important for the ease of end users. Before and after the ITS2 was accepted as an ideal barcode several researchers used different ITS2 primers for the identification of a diverse range of plants. Currently, in the literature, there are more than 20 sets of primers being mentioned for ITS2 (Table II). For a novice, it is very time-consuming and creates ambiguity in selecting the correct primer for their experiment. Therefore, in this manuscript, we did a comprehensive literature survey and identified a set of universal primers and validated them using in silico and in vitro analysis.