Abstract
Cinnamomum osmophloeum, an indigenous species of Taiwan, can be utilized for valuable products such as a food, a spice and a traditional Chinese medicine. This study compares the ribosomal DNA (nr DNA) internal transcribed spacer (ITS) sequence of C. osmophloeum to that of several other species with similar external morphology, such as Cinnamomum burmannii, Cinnamomum insularimontanum, Cinnamomum macrostemon and Cinnamomum subavenium. Phylogeny of ITS sequences shows that C. osmophloeum is more closely related to C. burmannii than the other species, while C. insularimontanum, C. macrostemon, and C. subavenium are phylogenetically relevant to each other. By comparing ITS sequence between C. osmophloeum and C. burmannii, specific primers were designed for the multiplex-PCR to differentiate them. Based on ITS sequence differences, all tested Cinnamomum spp. can be properly authenticated. A 125 bp band specific for C. osmophloeum and a 204-bp C. burmannii-specific band were successfully amplified by polymerase chain reaction (PCR) using the respective primers described above. The two species then can be identified at the molecular level according to the sizes of their respective PCR products as determined by gel electrophoresis. Key words: Cinnamomum osmophloeum, internal transcribed spacer (ITS), phylogenetic, multiplex- polymerase chain reaction (PCR).
Highlights
There are approximately 250 species in the genusCinnamomum
Specific primers were synthesized after comparing variations in the ITS2 sequences between the indigenous C. osmophloeum and exotic C. burmannii, and they were successfully used to differentiate the two species at the molecular level in a timely fashion
Primer pairs set specific for C. osmophloeum and C. burmannii were designed based on a single nucleotide difference found after comparing their ITS2 sequence (Figure 2)
Summary
Subtropical areas and Australia (Liu et al, 1994). Abbreviations: nr DNA, Ribosomal DNA; ITS, internal transcribed spacer; CTAB, cetyl trimethyl ammonium bromide; PCR, polymerase chain reaction; NCBI, National Center for Biotechnology Information; NJ, neighbor-joining method; MP, maximum parsimony; BLAST, basic alignment search tool. Presence and arrangement of bud scales and the presence of trichomes on the bud scales, inflorescence and perianth, are used as common morphological diagnostic characters for the species delimitation within the Cinnamomum genus (Liu et al 1994; Liao, 1996). As the two species are similar in appearance, they are not differentiated by their phenotypes, which often lead to economic losses because of planting errors by forest farmers. Various types of DNA markers provided by modern molecular biology can be used for delimitation of species that are not distinguished based on morphology. This study uses nuclear 5.8S rRNA and the ITS sequences as genetic markers to investigate the differences and phylogenetic relationships among C. osmophloeum and related similar species, including C. osmophloeum, C. insularimontanum, C. macrostemon, C. subavenium and C. burmannii. Specific primers were synthesized after comparing variations in the ITS2 sequences between the indigenous C. osmophloeum and exotic C. burmannii, and they were successfully used to differentiate the two species at the molecular level in a timely fashion
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