Organelle Genome Analysis And Identification Of Six Bidens Medicinal Plants

Plants of Bidens L.are annuals or perennials of Asteraceae.A total of ten species in the genus,and five of them are medical plants recorded in Guangdong,Guangxi,Shandong and other local standards of Chinese medicinal materials.Bidens biternate of this genus is one of the medicinal materials of 999 Ganmaoling.However,there are some problems in records about these medicinal plants of these local standards,for example,the scientific name is not consistent with their Chinese name and the records in flora of China.Bidens plants have similar morphologies and chemical components,so it is difficult to identify them by traditional identification methods.These problems seriously hinder the development of Bidens medicinal plants.Therefore,it is an urgent need to develop an accurate and effective method to identify the medicinal plants of Bidens.The chloroplast genomes are highly conserved in gene order and content,and they are thus ideal research models for the study of specific barcode and super barcode,species identification and evolution.The structure of mitochondrial genome in higher plants is more complex than chloroplast genome,but ITS/ITS2 gene composition is relatively conserved,which can be used for the study of genetic evolution of plants.In this study,DNA barcodes were used to identified Bidens medicinal plants.Illumina sequencing was used to sequence the chloroplast and mitochondrial genomes of six Bidens medicinal plants and the complete organelles genomes were assembled,annotated and analyzed.Specific barcodes were screened and ML phylogenetic tree were constructed based on the chloroplast genome to identified these medicinal plants.The results obtained in this study could provide valuable genetic resource for species identification,resource conservation and phylogenetic relationship analysis of the Bidens medicinal plants.The main results of this study are as follows:1.Comparative and identification analysis of Bidens medicinal plants by chloroplast genomes(1)ITS/ITS2 and psbA-trnH sequences were used to identified six Bidens medicinal plants(B.biternate,B.bipinnata,B.pilosa var.pilosa,B.pilosa var.radiata,B.parviflora and B.tripartita).A total of 21 samples of these six species were amplified and sequenced by sanger sequencing.The K2P genetic distance of intraspecific and interspecific of ITS/ITS2 and psbA-trnH sequences within these species were similar to each other.NJ phylogenetic tree showed that different species of Bidens clustered in one branch.Therefore,ITS/ITS2 and psbA-trnH sequences could not identify the six medicinal plants of Bidens.(2)The complete chloroplast genomes of 12 samples of B.biternate,B.bipinnata,B.pilosa var.pilosa,B.pilosa var.radiata,B.parviflora and B.tripartite were typical circular structure containing a pair of inverted repeats(IRs)separated by the large single copy(LSC)and the small single copy(SSC)regions.Chloroplast genomes ranged from 150,489 bp to 151,635 bp in length.The GC contents in the whole chloroplast genomes is 37.5%,which was highly conserved in Bidens species.A total of 130 genes,including 85 protein-coding genes,37 tRNAs and eight rRNAs were annotated,17 of which were duplicated in the IRs.The number of SSRs ranged from 60 to 71 in the 12 chloroplast genomes,which was not exactly the same in one species.The codon usage frequency was estimated and most of the amino acid codons show A/T base preferences.The relative synonymous codon usage(RSCU)value increased with the increase in the quantity of codons which coded for a specific amino acid.(3)The comparative analysis showed that the chloroplast genomes of six Bidens species were highly conserved,and the coding-gene sequences were more conserved than the noncoding sequences.Co-linear and homology analysis showed that the sequences of B.biternate and B.bipinnata are highly homologous,and B.pilosa var.pilosa and B.pilosa var.radiata are highly homologous.(4)Seven specific barcodes were screened base on the comparison of 12 complete chloroplast genomes,and four specific barcodes(ndhA,accD,clpP and ccsA-ndhD)were successfully amplified in these six species with 31 samples.Three specific barcodes(ndhA,accD and ccsA-ndhD)could identify B.parviflora and B.tripartite,and one specific barcodes(clpP)could not identify any species.Therefore,the specific barcodes were not suitable for the identification study of the six Bidens species.The result of ML phylogenetic tree constructed by the complete chloroplast genomes showed that six Bidens species clustered in a clade.Three B.biternate samples clustered in one branch,and four B.bipinnata clustered in one branch.B.pilosa var.pilosa and B.pilosa var.radiata clustered in one branch,which supported the result of Flora of China that the two species merged into one species.Therefore,the complete chloroplast genome could be used as a super barcode to identify six Bidens species.2.Mitochondrial genome analysis of Bidens medicinal plantsIn this study,the mitochondrial genomes of six Bidens species were analyzed for the first time.The length of complete mitochondrial genomes was between 183 kb and 216 kb.The GC contents in the whole mitochondrial genomes was approximately 45%and the number of coding sequence was similar among these six species.Apart from B.biternate and B.bipinnata,the number and type of SSRs and long repeats detected in mitochondrial genomes were different in the other four species.ML phylogenetic tree was constructed based on the published mitochondrial genomes of Asteraceae plants and the mitochondrial genomes obtained in this study.The results showed that B.biternate and B.bipinnata clustered in one branch,and B.pilosa var.pilosa and B.pilosa var.radiata clustered in one branch.Six Bidens species were not clustered in a clade.So,it is not feasible to identify Bidens medicinal plants by using the mitochondrial genome sequence.This study enriched the mitochondrial genome database of higher plants and was benefit for mitochondrial genome analysis of Asteraceae medicinal plants.The results of this study showed that the two DNA barcodes(ITS/ITS2 and psbAtrnH),special barcodes based on complete chloroplast genome and mitochondrial genome could not be used to identified these six Bidens species.The complete chloroplast genome could be used as super barcode to identified these six Bidens species.The results could enrich the organelle genome database of Asteraceae medicinal plants and provide foundation for molecular identification of Asteraceae plants.