Forensically informative nucleotide sequencing (FINS) is a molecular approach to identify biological materials based on DNA sequences. It focuses on the clustering of samples with reference species through an analysis of the phylogenetic relationship among the species. The choice of loci is critical to the successful use of FINS for identification. In this study, we investigated the influence of six loci (ITS, matK, rbcL, rps16, trnH-psbA and trnL-trnF) on the application of FINS for the identification of traditional Chinese medicine as demonstrated in Baihuasheshecao, a traditional anti-tumor herb derived from Hedyotis diffusa willd. A total of 36 plant samples of Hedyotis species and eight samples of Baihuasheshecao commodities were collected from Hong Kong, Macao, Taiwan, mainland China and the USA. Internal transcribed spacers (ITS) sequences displayed the highest rate of formation of single species cluster (89%), followed by rps16 (78%), matK (74%), trnL-trnF (72%), trnH-psbA (67%) and rbcL (61%). All the loci could discriminate H. diffusa from the common substitute species H. corymbosa, H. pinifolia and H. tenelliflora. FINS identification based on ITS, trnH-psbA and trnL-trnF revealed that five Baihuasheshecao commodities were genuine herb derived from H. diffusa, while the other three commodities were substitutes derived from H. corymbosa. MatK, rbcL and rps16 could not be amplified from all commodity samples and were thus inapplicable for FINS identification of dried the samples. In conclusion, the success of FINS identification depends on the DNA loci as they show different abilities to differentiate genuine species from substitutes, to discriminate closely related species and to generate quality sequences. In this study, the eligibility of DNA locus for FINS identification of Baihuasheshecao commodities, in descending order, is ITS > trnL-trnF > trnH-psbA > rps16 > matK > rbcL.

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