Molecular identification of Hippocampus trimaculatus and its adulterants based on DNA barcoding / 中草药

ABSTRACT

Objective: The aim of present study was to establish a DNA barcoding datebase of COI, 16 S rRNA and ATP6 sequences from Hippocampus trimaculatus and identify H. trimaculatus and other adulterants quickly and accurately. Methods: Total genomic DNA was isolated using the muscle tissue from tail of H. trimaculatus. The DNA barcoding genes of COI, 16 S, and ATP6 sequences were amplified by universal primers and PCR products were sequenced from both directions. Sequences assembly and consensus sequence generation were performed using the Codon Code Aligner V4.2. Sequence alignment was performed by using Clustal X software. The Kimura 2-Parameter (K2P) distances were calculated using the MEGA5.0. Identification analyses were performed using neighbor-joining (NJ) methods. Results: The length of the measured sequences of mitochondrial COI, 16 S, and ATP6 were 649 572 and 603-605 bp, respectively. The number of intraspecific variation sites of three genes was 8 bp, 4 bp, and 15 bp, respectively. The average intraspecific K2P genetic distance of COI, 16 S, and ATP6 were 0.002, 0.001, and 0.006. The genetic variation among H. trimaculatus was obviously less than the interspecific genetic variation. The NJ tree showed that H. trimaculatus could be distinguished obviously from other Hippocampus species, which showed high monopholy. Conclusion: Our results indicated that COI, 16 S, and ATP6 sequences are effective DNA barcodes for the identification of H. trimaculatus species and its adulterants, with view to providing the basis data for molecular identification of animal medicinal materials, adulterants and near-source species, and contributing to the clinical medication safety of H. trimaculatus.