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[摘要]
目的 利用DNA条形码技术评估ITS2序列对海南大戟科植物的鉴定能力。方法 利用PCR测序法,对供试样本ITS2片段进行双向测序,所得序列经Codon Code Aligner拼接后,利用MEGA 6.0进行相关数据分析,构建邻接(NJ)树。利用TAXON DNA软件分析序列种内、种间变异并作barcoding gap分析。从ITS2数据库中获得供试样本的预测ITS2二级结构。结果 大戟科供试样本ITS2序列中变异位点与信息位点分别占总序列长度的84%和69.67%。供试各种在构建的NJ树中均各自成束,表现出单系性;基于ITS2序列的种内最大变异距离值0.0673;种间最小变异距离值0.1191,各ITS2二级结构种内无明显差异;种间存在明显差异。结论 所得ITS2条形码序列对海南大戟科植物鉴定能力强,可作为DNA条形码序列对海南大戟科植物进行快速鉴定。
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[Abstract]
Objective To evaluate the ability of ITS2 region as a DNA barcode for identifying the plants in Euphorbiaceae in Hainan. Methods The ITS2 of ribosomal DNA was amplified and sequenced by bidirectional sequencing of PCR products. Sequence assembly and consensus sequence generation were performed by using Codon Code Aligner. Phylogenetic study was performed using software MEGA 6.0 in accordance with Kimura-2-parameter (K2P) model, and the phylogenetic tree was constructed by using the Neighbor-joining (NJ) method. The ITS2 secondary structure was predicted using the ITS2 Database server. The distribution of intra-versus inter-specific variability was compared using DNA bar coding gaps. Results The variable sites and the informative sites were accounted for 84% and 69.67% of the ITS2 regions length. In the cluster dendrogram, all species showed monophyletic. The maximum intra-specific distance was 0.067 3, and the minimum inter-specific distance was 0.119 1. There was significant bar coding gap between intra-specific and inter-specific variation. To compare the ITS2 secondary structure, we noticed that it had no obvious distinguishment of intra-specific, while had obvious distinguishment of inter-specific. Conclusion ITS2 sequence can be used as a DNA barcode for the rapid identification of the plants in Euphorbiaceae in Hainan.
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[基金项目]
海南省2013自然科学基金项目(813190)