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. 2015 Sep 11;16(9):21975-88.
doi: 10.3390/ijms160921975.

Molecular Identification of Dendrobium Species (Orchidaceae) Based on the DNA Barcode ITS2 Region and Its Application for Phylogenetic Study

Shangguo Feng  1   2 , Yan Jiang  3 , Shang Wang  4 , Mengying Jiang  5 , Zhe Chen  6 , Qicai Ying  7 , Huizhong Wang  8
Affiliations

Molecular Identification of Dendrobium Species (Orchidaceae) Based on the DNA Barcode ITS2 Region and Its Application for Phylogenetic Study

Shangguo Feng et al. Int J Mol Sci. .

Abstract

The over-collection and habitat destruction of natural Dendrobium populations for their commercial medicinal value has led to these plants being under severe threat of extinction. In addition, many Dendrobium plants are similarly shaped and easily confused during the absence of flowering stages. In the present study, we examined the application of the ITS2 region in barcoding and phylogenetic analyses of Dendrobium species (Orchidaceae). For barcoding, ITS2 regions of 43 samples in Dendrobium were amplified. In combination with sequences from GenBank, the sequences were aligned using Clustal W and genetic distances were computed using MEGA V5.1. The success rate of PCR amplification and sequencing was 100%. There was a significant divergence between the inter- and intra-specific genetic distances of ITS2 regions, while the presence of a barcoding gap was obvious. Based on the BLAST1, nearest distance and TaxonGAP methods, our results showed that the ITS2 regions could successfully identify the species of most Dendrobium samples examined; Second, we used ITS2 as a DNA marker to infer phylogenetic relationships of 64 Dendrobium species. The results showed that cluster analysis using the ITS2 region mainly supported the relationship between the species of Dendrobium established by traditional morphological methods and many previous molecular analyses. To sum up, the ITS2 region can not only be used as an efficient barcode to identify Dendrobium species, but also has the potential to contribute to the phylogenetic analysis of the genus Dendrobium.

Keywords: DNA barcode; Dendrobium; ITS2; genetic relationship; species identification.

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Figures

Figure 1
Figure 1
Relative distribution of inter-specific divergence between congeneric Dendrobium species and intra-specific variation in the ITS2 region using K2P genetic distance.
Figure 2
Figure 2
The heterogeneity and separability for individual taxa of ITS2 based on 64 Dendrobium species by TaxonGap. The left side shows the complete list of Dendrobium species used in this study, including sequences we generated and those retrieved from GenBank (see Table 3 and Table S1). The right side depicts the within species heterogeneity (presented as light gray horizontal bar) and between-species separability (presented as dark grey horizontal bar) values (calculated by using similarity matrix for biomarker ITS2) with different OTUs (Operational Taxonomic Units) as matrix rows for ITS2. The names of the closest relatives (the taxon with the smallest separability) are listed at the right side of the dark grey bar.
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