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Abstract

Water chestnut is an aquatic species belonging to Lythraceae family, widely valued for its medicinal and nutritional properties. DNA barcoding is an efficient technique which requires minimal tissue sample for species recognition. The aim of this work was the precise identification and phylogenetic assessment of Water chestnut based on ITS2 DNA barcode. DNA was isolated using the GeneJet plant genomic DNA kit and quantified via a Nano - drop microvolume spectrophotometer, and assessed for quality by 1% agarose gel electrophoresis. The nuclear molecular marker, ITS2 was amplified through for PCR. The ITS2 amplicons were purified and subjected to Sanger sequencing. The sequence was then aligned using Multiple Sequence Alignment (MSA) to identify conserved regions. This conserved ITS2 region was used for species identification through NCBI’s BLAST, confirming the species as Trapa natans L. by achieving a 99.41% correct identification rate. Phylogenetic tree based analysis was performed using MEGA6 with the ITS2 barcode, revealing a close relationship between Trapa natans L. Trapa acornis Osbeck., Trapa jeholensis Nakai., Trapa natans L. var. Bispinosa (Roxb.) Makino and Trapa natans L. var. Quadrispinosa (Roxb.) Makino, indicated by a branch length of 0.0000 at their nodes. This confirms ITS2 as a powerful nuclear barcoding region for identifying Trapa species and its utility for analysing and managing Trapa germplasm collections due to its biparental inheritance. But ITS2 does not elucidate cytoplasmic inheritance patterns due to lack of complementary cpDNA marker data. Future studies should employ “Integrative Taxonomy”, combining ITS2 data with chloroplast markers to detect natural hybridization and maternal lineages

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