Abstract
BackgroundInternal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns.Methodology/Principal FindingsIn this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level.ConclusionsIdentification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.
Highlights
The study of evolutionary novelty focuses on morphological traits [1] as well as genomic and proteomic data [2], especially molecular sequence data [3,4,5,6,7,8,9]
Intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases
Our results strongly suggest that the intra-genomic internal transcribed spacer 2 (ITS2) variants can reflect the phylogenetic history of species and genera
Summary
The study of evolutionary novelty focuses on morphological traits [1] as well as genomic and proteomic data [2], especially molecular sequence data [3,4,5,6,7,8,9]. The internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA cistron is one of the most frequently used markers for phylogenetics, diagnostics and DNA barcoding [10,11,12,13,14,15,16,17,18]. There are hundreds of copies of ITS2 with potentially dozens of different sequences, so sequence comparisons involving just one or a few PCR-amplified ITS2 sequences from each species of interest may lead to inaccurate or misleading results [20] This largely overlooked intra-genomic divergence of ITS2 sequences can complicate the use of this marker in phylogenetic and barcoding applications.
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