Comparative Chloroplast Genomics And DNA Barcoding Of Medicinal Plants Of Potentilla S.str. In China

The genus Potentilla L.belongs to family Rosaceae Juss.,subfamily Rosoideae(Juss.)Arn.,tribe Potentilleae Sweet.The genus Potentilla constitutes approximately 485 species worldwide.Potentilla sensu stricto includes Potentilla,Horkelia Cham.et Schltdl.,Horkeliella(Rydb.)Rydb.,Ivesia Torr.et A.Gray.and Duchenea Sm.The medicinal plants of Potentilla have anti-inflammatory,anti-viral,hypoglycemic,anti-ulcer,anti-tumor,dampness removing and analgesia and analgesic,anti-hyperlipidemic,anti-oxidative and other pharmacological effects.There are 20 species and four varieties of medicinal plants of Potentilla s.str.in China.In view of the position and role of medicinal plants of Potentilla s.str.in traditional Chinese medicine,it is necessary to study its chloroplast genome and molecular identification.In this study,based on the Illumina Novo Seq PE150 paired-end sequencing platform,chloroplast genomes of 16 populations of 10 species(including variety)of the medicinal plants of Potentilla s.str.and Drymocallis saviczii were obtained.The chloroplast genomes of 17 populations obtained in this study and the chloroplast genomes of 17 related populations downloaded from the NCBI database were integrated,and the medicinal plants of Potentilla s.str.in China were studied from the perspective of comparative chloroplast genomes and DNA barcoding.This study is helpful to the species identification and phylogenetic relationship research of medicinal plants of Potentilla s.str.The screened candidate DNA barcoding and chloroplast genomes Ultrabarcoding can be used for the identification of medicinal plants of Potentilla s.str.,which is beneficial to the clinical differentiation and use and development and application of the medicinal plants of Potentilla s.str.The main contents and results of this study are as follows:1.The chloroplast genomes of Potentilla s.str.all showed a typical tetrad structure,including a large single copy region,a small single copy region and two inverted repeat regions of equal length.The chloroplast genomes of Potentilla s.str.all encoded 129 genes,including 84 protein-coding genes(PCG_S),37 transfer RNA(t RNA)genes and 8 ribosomal RNA(r RNA)genes.The chloroplast genome sequences of Potentilla s.str.had little difference in length(155,044-156,446bp),and the GC contents were 36.9%-37.0%.In addition,there was no inversion and gene rearrangement in the chloroplast genome of Potentilla s.str.,and there was no obvious contraction or expansion in the IR region.2.Based on the chloroplast genome sequences of Potentilla s.str.,nine fragments(rps4-trnT,trnR-atpA,trnH-psbA,rps2-rpoC2,rpl32-trnL,petA-psbJ,trnG-trnR,trnT-trnL and psaJ-rpl33)were developed as hypervariable molecular markers of Potentilla s.str.The nine fragments were all intergenic regions,of which the rpl32-trnL fragment was located in the SSC region,and the remaining eight fragments were located in the LSC region.The obtained chloroplast genome sequences of 16 populations of 10 species(including variety)could be used as Ultrabarcoding for the identification of medicinal plant species of Potentilla s.str.3.Phylogenetic trees were reconstructed for Potentilla s.str.and related taxa based on the maximum likelihood method and Bayesian inference.The topologies of maximum likelihood tree and Bayesian tree showed a high consistency,and the outgroups were separated from Potentilla s.str.with a high support.Potentilla s.str.contains six large clades,including Himalayan clade,Alba clade,Reptans clade,Fragarioides clade,Ivesioid clade and Argentea clade.4.The identification ability of matK,rbcL and trnH-psbA fragments as standard barcoding in 30 populations of 22 species(including varieties)of Potentilla s.str.was analyzed.According to the sequence diversity and interspecific and intraspecific genetic distance,trnH-psbA fragment has the shortest length but the highest variability,and has the characteristics of ideal DNA barcoding for identifying medicinal plants of Potentilla s.str.According to the phylogenetic analysis,the NJ tree cluster based on matK is the most ideal,then followed by NJ tree cluster base on rbcL,and the identification ability of trnH-psbA fragment is the lowest.Compared with the identification ability of a single fragment,the identification ability of the combined barcoding(matK+rbcL?matK+trnH-psbA?rbcL+trnH-psbA and matK+rbcL+trnH-psbA)has more dominant.5.The identification ability of nine candidate fragments(trnG-trnR,trnH-psbA,psaJ-rpl33,trnR-atpA,rps2-rpoC2,trnT-trnL,petA-psbJ,rpl32-trnL,and rps4-trnT)as specific barcoding in 30 populations of 22 species(including varieties)of Potentilla s.str.was analyzed.According to the sequence diversity and interspecific and intraspecific genetic distance,trnG-trnR fragment has the shortest length but the highest variability.The tree-building results showed that petA-psbJ,trnT-trnL and rpl32-trnL had the best identification ability,and their identification was better than rbcL and trnH-psbA,while the identification ability of trnG-trnR,trnH-psbA and rps4-trnT was poor.The results fully demonstrate the importance of considering fragment length while ensuring variability when screening hypervariable fragments.The three fragments with the best performance were analyzed jointly,and the combined fragment(petA-psbJ+trnT-trnL?petA-psbJ+rpl32-trnL?trnT-trnL+rpl32-trnL and petA-psbJ+trnT-trnL+rpl32-trnL)has more dominant in identification ability.6.The identification ability of chloroplast genome sequence as Ultrabarcoding in 30 populations of 22 species(including varieties)of Potentilla s.str.was analyzed.The sequence length,variation sites and parsimony information sites of Ultrabarcoding are all larger than standard barcoding and specific barcoding.The results show that Ultrabarcoding can provide more abundant genetic variation information,which can not only improve the identification ability of Potentilla s.str.,but also have higher reliability.