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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 DNA barcode sequences of 6 species of Aloe. Methods The genomic DNA of 18 samples from six species of Aloe was extracted by kit method. ITS2 and chloroplast psbA-trnH, rbcL and matK were amplified by universal primers and sequenced by Sequencher 4.1 .4 software for sequencing the peak map proofing stitching, the application of MEGA 5.0 software analysis of the characteristics of different candidate sequences to calculate the species within and between species Kimura 2-Parameter (K2P) genetic distance, compared with intraspecific and interspecies variation size The sequence barcoding gap was evaluated, and the system clustering tree was constructed by neighbor-joining (NJ). Results A total of 72 sequences were obtained. The sequences of ITS2, psbA-trnH, rbcL and matK were all 18, and the success rate of sequencing was 100%. The aligned sequences ranged in length from 255 bp to 723 bp with a GC range of 30.6% to 68.2%. The maximum intraspecific distance of psbA-trnH gene sequence was less than the minimum interspecific genetic distance. There was some overlap between interspecific variation and interspecies variation of barcoding gap, ITS2, rbcL and matK sequences, and no obvious barcoding gap. The developmental tree based on psbA-trnH sequence can completely distinguish six varieties of Aloe, while the other three sequences have fuzzy identification in distinguishing these Aloe species. Conclusion The DNA barcoding technique can accurately identify Aloe species, and the psbA-trnH sequence can be used as a preferred sequence to identify Aloe species.