Phylogenetic Analysis Of Angelica Sinensis And Its Relatives Based On Chloroplast Genome Sequence

Objective: Angelica sinensis(Oliv.)Diels belongs to Angelica L.of Apioideae(Apiaceae)in classical taxonomy.Studies based on modern molecular systematics show that A.sinensis belongs to the Sinodielsia Clade,which is far from most species of Angelica in classical taxonomy.The species of Sinodielsia Clade are various and their sources are complex.The systematic relationship of species in the clade is still unclear.The purpose of this study is to identify the related species of A.sinensis in the Sinodielsia Clade,analyze the phylogenetic relationship between A.sinensis and its related species,and find potential molecular markers that can be used for the phylogenetic evolution and species identification of the Sinodielsia Clade and even the subfamily Apioideae.Methods: Based on the structural characteristics of chloroplast(CP)genome and its advantages in phylogenetic research,39 species belonging to 9 genera within the Sinodielsia Clade and related clades were selected for DNA sequencing,CP genome assembling and annotating.Then we analyzed the basic characteristic information of CP genome.Using the newly assembled CP genome data in this study,combined with the published CP genome data of Sinodielsia Clade and related clades species in Gen Bank database,the ML phylogenetic tree based on the whole CP genome and common CDS sequence was constructed.The phylogenetic relationship between Sinodielsia Clade was analyzed to identify the species closest to A.sinensis.Further comparative analysis of CP genome of Sinodielsia Clade was carried out to analyze the characteristics and screen the highly variable region of genome.Result: We sequenced,assembled and annotated the CP genomes of 39 Sinodielsia Clade and related clades(All reported for the first time).Through the analysis of the basic structural characteristics,it was found that 39 CP genomes all showed a typical circular structure,which was composed of LSC,SSC and two IR regions.The total length of the genome is 145,335 ～ 165,147 bp,and the number of coding genes is 126 ～ 138,including83 ～ 93 CDS genes,35 ～ 37 t RNA genes and 8 r RNA genes.In this study,a total of 2734 SSRs were detected in 39 CP genomes,of which 1577 were Mononucleotide,and the number of Pentanucleotide and Hexanucleotide were relatively small;969 Forward repeats,930 Palindromic repeats,52 Reverse repeats and 46 Complement repeats were also detected.Phylogenetic analysis showed that 19 species from 10 genera of Apiaceae clustered into the Sinodielsia Clade.Previously reported 7 species from 6 genera were excluded from the Sinodielsia Clade,and 3 species were clustered into the Sinodielsia Clade firstly.There are two subclades in the Sinodielsia Clade.Nine A.sinensis sequences were divided in the two subclades of the Sinodielsia Clade,respectively.In the Sinodielsia Clade,the species closest to A.sinensis are Cnidium officinale Makino,Conioselinum chinense Hoffm.,Ligusticum sinensis Oliv.,Lig.chuanxiong Hort.,Lig.jeholense(Nakai&Kitag.)Nakai&Kitag,Lig.tenuissimum(Nakai)Kitag,A.omeiensis Yuan et Shan,A.paeoniifolia Yuan et Shan,A.ternata Regel et Schmah.,and A.multicaulis Pimenov.The IR region of the CP genome of the Sinodielsia Clade had certain expansion and contraction.The variation of the non-coding region was greater than that of the coding region,and the variation of the LSC and SSC region was greater than that of the IR region.We have detected six highly variable regions of rbc L–acc D,ycf4–cem A,pet A–psb J,ycf1–ndh F,ndh F–rpl32,and ycf1 genes in the genome of the Sinodielsia Clade species,among which ndh F–rpl32 and ycf1 genes also show high variability in all related species of the Sinodielsia Clade.These regions,especially ndh F–rpl32 and ycf1 genes,can be preferentially developed as potential molecular markers for specific DNA barcode.It will be used for future research on the phylogeny and species identification of the Sinodielsia Clade and even Apioideae.Conclusion: The total length of 39 CP genome first reported in this study is 145335 ～165147 bp,and the number of coding genes is 126 ～ 138,including 2734 SSRs and 1997 long repeats.These data enriches the CP genome database of Apiaceae and provides a data base for the molecular systematics in the future.A.sinensis is closely related to Cni.officinale,Con.chinense,Lig.sinensis,Lig.chuanxiong,Lig.jeholense,Lig.tenuissimum,A.omeiensis,A.paeoniifolia,A.ternata,and A.multicaulis.These species provide available genetic resource information for the future variety improvement and breeding of A.sinensis.Six highly variable regions of the CP genomes were detected in the Sinodielsia Clade and related clades,which provided reference information for the development of DNA barcode in Sinodielsia Clade even Apiaceae.