Molecular Structure And Identification Analysis Of Chloroplast Genomes Of Ligularia And Ephedra Herbs

Toxic plants generally refer to plants that have harmful effects on people and livestock.Toxic plants are rich in toxic components,such as alkaloids and glycosides,which are hazardous to the nervous and respiratory system.In fact,many toxic plants are also medicinal plants with important medicinal and economic value.The genera of Ligularia Cass.(Asteraceae)and Ephedra Tourn ex Linn.(Ephedraceae)in this study have a long history of medicinal use.However,some plants of Ligularia contain pyrrolizidine alkaloids,which are harmful to human and animal health and are involved in metabolic toxification in the liver.Overdose use of Ephedra plants containing ephedrine could cause excitation in the central nervous system,leading to poisoning.Accurate identification of them can ensure drug safety and avoid unnecessary losses.Here,we report the complete chloroplast(CP)genomes of six Ligularia species and three Ephedra species obtained through high-throughput Illumina sequencing technology.This study provides invaluable data for species identification,allowing for future studies on phylogenetic evolution and safe medical applications of Ligularia and Ephedra.1.Chloroplast genomes of six Ligularia speciesLigularia comprises about 140 species of perennial herbs.These herbal medicines are usually used as Ziwan or Shanziwan and more than 30 species of this genus have long been used in folk medicine in China.Morphological features and the universal DNA barcodes were both not ideal to identify Ligularia species.Thus,CP genomes of Ligularia intermedia Nakai,Ligularia jaluensis Kom.,Ligularia mongolica(Turcz.)DC.,Ligularia hodgsonii Hook.,Ligularia veitchiana(Hemsl.)Greenm.,and Ligularia fischeri(Ledeb.)Turcz.were sequenced and analyzed.(1)The six Ligularia CP genomes showed typical circular tetramerous structure including a large single-copy(LSC)region,a small single-copy(SSC)region and two inverted repeats(IRa and IRb)regions.The sizes of CP genomes ranged from 151,118 bp to 151,253 bp.The sizes of SSC,LSC and IR regions were approximately 18.2 kb,83 kb and 24.8 kb,respectively.The GC content of each CP genome was 37.5%.(2)Each CP genome of six Ligularia species encoded 134 genes,including 87 protein-coding genes,37 tRNA genes,eight rRNA genes,and two pseudogenes(ycf1 and rpsl9 genes).(3)The CP genomes of six Ligularia species contained different types and lengths of long repeats.They all contained four types of simple sequence repeats(SSRs):mononucleotide(the proportion of all SSRs:56,6-60.7%),dinucleotide(11.5-13.2%),trinucleotide(9.3-9.8%)and tetranucleotide(18.0-21.6%).SSRs were mainly distributed in the non-coding region of the single-copy regions.(4)The structure and composition of the six Ligularia CP genomes are highly similar.Like most Asteraceae species,each CP genome of six Ligularia species included a small 3.4 kb inversion within a large 23 kb inversion in the LSC region.The mVISTA showed that no obvious hypervariable region was screened to identify the six species.An alignment of the six whole CP genomes found that the CP genome was expected to be used as a super-barcode for identification of Ligularia species.The maximum likelihood(ML)tree of the six Ligularia species and other related species showed that six Ligularia species formed a single branch with support value of 100%.2.Chloroplast genomes of three Ephedra speciesEphedra belongs to Gymnospermaae and comprises approximately 40 species.They are distributed in arid and desert regions of Asia,Southeastern Europe,Northern Africa and America.Ephedrae Herba and Ephedrae Radix et Rhizoma(Mahuang)have been used as Chinese herbal medicines for thousands of years.Morphological resemblance amongst species causes difficulty in identifying the three original species(Ephedra sinica Stapf,Ephedra intermedia Schrenk ex Mey.,and Ephedra equisetina Bge.)of Ephedra herbs in the Chinese Pharmacopoeia,even the common DNA regions.Therefore,we sequenced the CP genomes of the three Ephedra species and screened specific DNA regions to identify Ephedra species.(1)The three complete CP genomes of Ephedra showed four-part annular structures.The sizes of CP genomes were 109,550 bp(E.sinica),109,667 bp(E.intermedia),and 109,558 bp(E.equisetina),respectively.The sizes of LSC,SSC and IR regions were about 59.9 kb,8 kb,and 20.7 kb,severally.The GC contents of three CP genomes was about 37%.The GC content of LSC and IR regions was 34.2%and 42%,individually.The GC content in the SSC regions was 27.3-27.8%.(2)Each CP genome of the three Ephedra species encoded 118 genes,including 73 protein-coding genes,37 tRNA genes and 8 ribosomal RNA genes.Compared with CP genomes of Nicotiana L.,the three CP genomes had no NADH(Nicotinamide adenine dinucleotide)dehydrogenase genes,rpsl6,rpl23,rpl32,ycf15 and accD genes.But the three CP genomes contained chlB,chlL,chlN genes,and photosystem ? related genes named psbL,psbM,psbN,psbT and psbZ.(3)The numbers of SSRs in the CP genomes of E.intermedia,E.sinica,and E.equisetina were 55,62 and 59,respectively.There were 37,4,4,and 10 SSRs in E.intermedia in LSC,IRa,IRb,SSC regions,severally.There were 39,5,5,and 13 SSRs in E.sinica in LSC,IRa,IRb,SSC regions,respectively.There were 41,4,4,and 10 SSRs in E.equisetina in LSC,IRa,IRb,SSC regions,individually.The CP genomes of three Ephedra species contained different types and lengths of long repeats.(4)Eleven high-variation regions,including two genes(ycf3 and rpl2)and nine intergenic spacers(psbZ-trnG,petN-rpoB,trnR-trnM,psbJ-rpl20,clpP-psbB,rrnl6-trnl,rps15-ccsA,ycfl-rps15 and trn V-rps12)were screened through mVISTA to be potential DNA barcodes for identifying Ephedra species.The ML and maximum parsimony(MP)trees constructed by 53 common protein-coding genes showed that CP genomes were expected to be used for identification of Ephedra species.This study provides theoretical foundation for the safe use of Ephedra and the phylogenetic relationships of gymnosperms.