Identification of DNA Barcode Sequence and Genetic Relationship among Some Species of Magnolia Family

摘要

Background and Objective: Magnoliaceae is one of the arguable botanist families in classification by conventional taxonomic methods. DNA barcoding has been proposed as a powerful tool for identifying, confirming species and genetic relationship among species. Using DNA barcoding can overcome problems of morphological-based species identification. The objective of this study was to isolate, analyze and determine the DNA barcode regions for species identification and analysis of genetic relationship among some species of Magnolia family. Materials and Methods: The leaf specimens were collected and dried in situ with silica gel and then stored in -20°C freezer. Total DNA extraction and PCR amplification were performed with standard chemicals and Kit and these DNA fragments were sequenced by Sanger method. The data analysis was supported by software programs such as Mega6, BioEdit, GeneDoc. Results: In this study, four candidate DNA barcode sequences including mat K, rbc L, trn H- psb A and ycf 1b were isolated and sequence analysed. The result showed that their size were 714, 553, 509-514 and 909-962 bp, respectively. The result of genetic distance analysis showed that only trn H- psb A fragment is best discrimination between four studied plant species. The combinations of ycf 1b+ trn H- psb A , mat K +trnH-psbA or matK + ycf 1b+ trn H- psb A also were potential choices to be used as DNA barcode for these four species. The phylogenetic trees were built based on trn H- psb A sequences and the combinations above revealed the genetic relationship of these four species, in which Magnolia chevalieri (Dandy) V.S.Kumar was closer with Michelia braianensis Gagnep. than Michelia tonkinensis A.Chev and Michelia baillonii (Pierre) Finet and Gagnep. Conclusion: Among 4 candidate DNA barcodes in this study, the psb A -trn H region is the most efficient DNA barcode sequence. Furthermore the combinations ( mat K+ trn H- psb A, rbc L+ trn H- psb A, ycf 1b+ trn H- psb A, mat K+ ycf 1b+ trn H- psb A, mat K+ rbc L+ ycf 1b+ trn H- psb A) could be used as the DNA barcode for species identification and analysis of genetic relationship among some species of Magnolia family.