Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA

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.背景 核核糖体DNA（nuclear ribosomal DNA, nrDNA）的内转录间隔区（internal transcribed spacer, ITS）已是当前应用最为广泛的系统发育与DNA条形码标记之一。然而，该序列多拷贝的存在为其应用带来了复杂性，对基因组内变异进行精准表征是解决此类问题的关键所在。 研究方法与主要发现 本研究采用序列标签焦磷酸测序（sequence-tagged pyrosequencing）与全基因组分析技术，对178种植物的内转录间隔区2（internal transcribed spacer 2, ITS2）的基因组内变异特征进行了系统解析。研究发现，ITS2的突变事件频发，单个物种平均存在35个ITS2变异体；其中丰度最高的3种变异体平均可覆盖所有ITS2拷贝的91%。此外，我们在13个属中观测到不同同属物种共享完全一致的ITS2变异体。值得注意的是，跨属的不同物种间同样存在共享的ITS2变异体：尤为典型的是，在五加科（Araliaceae）中，太白山五加（Eleutherococcus giraldii）的一种次要ITS2变异体，与人参（Panax ginseng）的主要ITS2变异体序列完全一致。另外，DNA条形码缺口分析结果显示，基因组内的遗传距离显著小于种内或种间变异的遗传距离。在对5543个ITS2变异体逐一开展物种区分效率检测后，我们在物种水平上获得了97%的识别成功率。 结论 同属乃至跨属物种间共享相同ITS2变异体的现象，揭示了物种演化历史的复杂性，该现象可能与水平基因转移及祖先杂交事件相关。尽管多数物种的基因组内普遍存在多拷贝的ITS2变异体，但在大多数研究场景中，仅使用主要变异体即可满足系统发育构建与物种鉴定的需求。进一步纳入次要变异体，则可有效提升物种鉴定的分辨率。