目的 筛选出适合芦荟属6种植物的DNA条形码序列。方法 采用试剂盒法提取芦荟属6个品种基原植物的18份样本基因组DNA,应用通用引物扩增其核基因ITS2序列和叶绿体psbA-trnH、rbcL、matK基因序列并进行双向测序,采用Sequencher 4.1.4软件对测序峰图进行校对拼接,应用MEGA 5.0软件分析不同候选序列的特征,计算物种的种内、种间Kimura 2-Parameter(K2P)遗传距离,比较其种内、种间变异的大小,评估序列的条形码间距(barcoding gap),采用邻接法(neighbor-joining,NJ)构建系统聚类树。结果 共获得72条序列,ITS2、psbA-trnH、rbcL、matK基因序列各18条,测序成功率均为100%。比对后序列长度为255~723 bp,GC量范围为30.6%~68.2%。psbA-trnH基因序列的最大种内遗传距离均小于最小种间遗传距离,有明显的barcoding gap,ITS2、rbcL、matK序列的种内变异和种间变异有一些重叠,没有明显的barcoding gap。基于psbA-trnH序列的发育树能将芦荟属6个品种完全区分,而其他3个序列在区分这些芦荟属品种时存在模糊鉴定。结论 DNA条形码技术可以准确鉴别芦荟属植物,psbA-trnH序列可以作为鉴别芦荟属植物的优选序列。

Objective To screen out a proper DNA barcode for suitable of six species in Aloe L. Methods The genomic DNA from 18 samples of six species in Aloe L. was extracted by Plant Genomic DNA Kit, then nuclear ITS2 and the chloroplast psbA-trnH, rbcL, and matK of the samples were amplified using the universal primers and sequenced bidirectionally. The obtained sequences were assembled using Sequencher 4.1.4. The characteristics of four candidate DNA barcodes were analyzed and the Kimura 2-Parameter (K2P) distances were calculated based on the sequences using MEGA 5.0. DNA barcoding gaps were evaluated by the analysis of the intra- and inter-specific divergences. The phylogenetic trees were constructed on the basis of Neighbor-Joining method. Results Totally 72 sequences of species in Aloe L. were acquired altogether, with each of ITS2, psbA-trnH, rbcL, and matK regions having 18 sequences respectively. The successful sequencing rate was 100% of all the four candidate barcodes. The alignment length and GC contents ranged from 255 to 723 bp and 30.6% to 68.2%. The maximum K2P intra-specific genetic distances were much smaller than the minimum inter-specific genetic distance of all samples in terms of psbA-trnH sequence and thus forming into a prominent barcoding gap. Due to some overlaps of intra- and inter-specific variation, there was no obvious barcoding gap in the aspect of ITS2, rbcL, and matK sequences. The phylogenetic tree constructed by psbA-trnH sequence was capable to identify the six species of Aloe L. while there existed fuzzy identification as to the other three candidate barcodes. Conclusion DNA barcoding technology can identify species in Aloe L. accurately and psbA-trnH sequence can be the optimal barcode to identify the species of Aloe L.

广东省教育厅高等院校学科与专业建设专项资金项目(粤财教[2013]412号);广州中医药大学2014年度"薪火计划"资助项目(XH20140109)