Disorganized and chaotic collection of the Euphorbia plant species from the wild is one of the major reasons for its endangered status. According to CITES, the trade in Euphorbia royleana species is prohibited under Appendix II. However, the trade continues unabated as current identification methods do not discriminate between closely related species.
In the present study, a DNA barcoding method has been used to establish inter- and intra-specific divergences of both matK and rbcL regions by using pairwise genetic distance measurement methods for evaluating the maximum barcoding gap.
The matk and rbcL yielded a 100% amplification and sequencing success rate to distinguish closely related species of Euphorbia royleana unambiguously. The matk and rbcL showed average interspecific genetic distance divergence values of 0.031and 0.015, respectively. The maximum number of species-specific SNPs was observed in matK sequences at seven consecutive sites, which could distinguish Euphorbia royleana from closely related species.
The best candidate barcoding region to identify Euphorbia royleana was found to be matK with a single-locus barcoding approach. Furthermore, the species discrimination method was developed with the help of species-specific SNPs derived from the matK barcoding region to accurately authenticate Euphorbia royleana, and it provided 100% species resolution
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