| Forsythia suspensa belongs to Oleaceae in Lamialesa with a high medicinal value for fighting inflammation and virus.With rising standards of living and rapid increasing of demand,the traditional identification is poor in the market with fakes.Therefore,techniques of molecular biology are more popular for identification.DNA barcode was put forward by Herbert in 2003,in which rapid and accurate species identification and discovery were performed by using short and standard fragment of DNA sequences.In this paper,eighteen samples from different origins were collected for further analysis,and genomic DNA extracted from samples by using high salt low pH method.Five sequences,ITS2,psb-trnH,rbcL,ycf5 and matK,were amplified and sequenced.The PCR amplification,mutated sites and phylogenetic tree were assessed.The main result were as follows:1.Genomic DNA was isolated from eighteen species of Forsythia suspensa and its adulterants using high salt low pH method.2、The results of PCR showed one DNA fragment was amplified from ITS2,matK,ycf5,rbcL,psb-trnH of eighteen samples,respectively.Success rate of amplification of rbcL,psb-trnH,ITS2 were 100%.3.The sequences and mutated sites were analyzed by using MEGA6.0 for Forsythia suspensa and its adulterants.The results showed there are many mutated sites in psb-trnH from different origins,but no variation were observed inrbcL.The amount of variation in ITS2 and rbcL,the former was the largest and another one was the least according to identification of Forsythia suspensa and its adulterants.4.Phylogenetic tree what obtained from ITS2,rbcl,ycf5,matK,psb-trnH showed all samples were distributed to three groups.The different origins of Forsythia suspensa belonged to one group.Forsythia viridissima Lindl.and Forsythia mandshurica Uyeki belonged to another,Syringa oblata Lindl.belonged to the last group.Compared with the mitochondrial genome and nuclear genome,chloroplast genome has greater advantages in the analysis of plant genetic relationship,it has been proposed as super barcode for species identification by some scholars.In this study,the optimal method of extracting the Forsythia suspensa chloroplast DNA was selected,obtained the entire chloroplast genomes of Forsythia suspensa in Zezhou、Tongguan based on high-throughput sequencing data and maps of chloroplast genome,analyzed the characteristics of the chloroplast genome structure and structural differences of two kinds of Forsythia suspensa,searched simple sequence repeats,compared the basic characteristics of chloroplast genome and IR border region with four species in Lamiales,MP and ML phylogenetic tree were formed based on 68 shared coding-genes for a total of 10 samples of Oleaceae.The main results were as follows:1.The experiments respectively extracted Forsythia suspensa DNA by sucrose density gradient centrifugation method、high salt and low pH method、improved high salt and low pH method and DNase I method.The results showed that,the extraction of improved high salt and low pH method with good purity and a great number of intact chloroplast,thus it is the optimal extraction method.2、The Forsythia suspensa chloroplast genome were selected from genome sequencing and assembled,the chloroplast genome of Forsythia suspensa in Zezhou was found to be 156404 bp in length which included a pair of inverted repeats(IRs)of 25717 bp,separated by a small single-copy region(SSC)of 17761 bp and a large single-copy region(LSC)of 87159 bp.the chloroplast genome of Forsythia suspensa in Tongguan with the length of 156378 bp contained 25707 bp-long IR region,87130 bp-long LSC and 17834 bp-long SSC.A total of 133 predicted genes including 79 protein-coding genes,4 ribosomal RNA genes and 30 tRNA genes were identified.3.Based on a comparative analysis of chloroplast genomes IR regions from Forsythia suspensa in Zezhou、Tongguan and four species in Lamiales,ycfl pseudogene was observed in all species IR/SSC border,the overlapping phenomenon were found between ycfl and ndhF gene in Salvia miltiorrhiza、Olea europaea、Forsythia suspensa in Zezhou、Tongguan,the distance between trnH in LCS region and IR/SSC border was 10-23 bp.4、MP and ML phylogenetic tree were formed based on 68 shared coding-genes for a total of three genera 10 samples of Oleaceae,the results showed that MP phylogenetic tree what obtained from Olea europaea,Forsythia suspensa,Jasminum nudiflorum showed three species were distributed to three groups.The results of the chloroplast genome have an enormous potential in identifying species. |
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