Molecular identification of six species in Aloe L. based on DNA barcoding / 中草药

ABSTRACT

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.