DNA Barcoding Development In Pulsatilla(Ranunculaceae)

Pulsatilla(Ranunculaceae)consists of about 40 species mainly distributed the Northern Hemisphere.There are about 11 Pulsatilla species recognized in China.Pulsatilla plants have medicinal properties and contain numerous compounds,such as anemonin.However,the recognition and identification of wild Pulsatilla species is difficult due to the presence of complex morphological characters.DNA barcoding is a technique for rapid,accurate taxon identification using intraspecific specificity and interspecific diversity of one or a few short,standardized DNA region(s).At present,this technology has a development tendency from universal barcode(including single-locus DNA barcodes and candidate multi-locus DNA barcodes)to chloroplast genome ‘super-barcoding' and specific barcode.The rapid and effective identification of Pulsatilla plants by this technique has important theoretical significance and application value.In total,52 accessions representing 19 Pulsatilla species were involved in this study.This sample covered each of the three subgenera from Asia,Europe,and America.The methods of Sanger sequencing,Next-generation sequencing(NGS)and Scanning Electron Microscopy(SEM)were used to get the data of the four universal barcodes(ITS,matK,rbcL,trnH-psbA)sequences from 52 individuals,the sequences of chloroplast genome of 7 individuals and the leaves' micro-morphological characteristics of 8 species in Pulsatilla.This study is analyzed from three aspects: to evaluate the species identification capability of universal barcodes,to explore the resolution level of the combination of leaf micro-morphological characters and barcodes and comparison of chloroplast genomes and phylogenetic analysis.The objective of this study was to select the best barcodes and barcode combinations,to verify the systematic significance of micro-morphological characteristics of leaves as auxiliary barcodes and screening of barcodes for Pulsatilla based on chloroplast genome.Moreover,it is expected to provide a reference for the rapid identification of Pulsatilla.Primary results are as following:1.Evaluation results of universal barcode for Pulsatilla.(1)Sequence characteristics and success rate of amplification and sequencing: the amplification and sequencing success rates of ITS and trnH-psbA were high(100%),and were not affected by mutation site or indel.it was difficult to amplify rbcL and matK from the degraded DNA isolated from old herbarium specimens.the amplification and sequencing success rate may be increase by using additional pairs of primers(96.15%).(2)Pair-wise genetic(PWG)distance: the ITS sequence has the largest intraspecific and interspecific distance,and the interspecific difference of barcode combination rbcL+matK+trnH-psbA was most suitable for species identification.(3)Sequence similarity:among the four single barcodes,the ITS and trnH-psbA had the highest success rate(79.06%)for the correct identification of species.Among the combinations,matK+ITS performed the best(83.72%).(4).Species discrimination:the barcode combinations with high species resolution is rbcL+trnH-psbA(70.73%)and rbcL+matK+trnH-psbA(58.54%).Overall,we propose that the rbcL+matK+trnH-psbA combination and the ITS marker on its own are,respectively,the best multi-and single-locus barcodes for discriminating among species of Pulsatilla.2.Systematics significance of leaf micro-morphological characteristics.Based on ITS and barcode combination rbcL+matK+trnH-psbA,the Bayesian inference(BI)tree of Pulsatilla was constructed respectively.The results showed that the 19 species involved in the study were divided into three single branches,corresponding to the three subgenera(Subg.Kostyzewianae,Subg.Preonanthus,Subg.Pulsatilla)of the classical classification system of Pulsatilla.But within the Subg.Pulsatilla,there are differences in topological structure between the BI tree of ITS and rbcL+matK+trnH-psbA.This may be caused by incomplete lineage sorting,introgression,and/or hybridization.Combined with the analysis of leaf micromorphological characteristics of the subg.Pulsatilla,it was found that the characteristics of density and length of leaf epidermis trichomes,density and type of stomata,and contour of leaf epidermal cell had important systematic significance.It can be used to distinguish some closely related species in Pulsatilla.3.Phylogenetic analysis of chloroplast genome and screening of specific barcode in Pulsatilla.Seven chloroplast genome sequences of Pulsatilla were obtained by NGS.Phylogenetic trees were constructed by seven chloroplast genome which were obtained in this study and four species chloroplast genomes which have been published.The results showed that these eleven species were divided into three groups and had extremely high support,which proved that chloroplast genome could be used as a super barcode for species identification of Pulsatilla.By sliding window analysis of chloroplast genome sequence,it is found that there are 8 variation hotspots,including non-coding regions rps16-matK,rps4-rps16,ndhC-trnV,psbE-petL,ndhD-ccsA,ccsA-ndhF and coding regions ndhF,ycf1.These markers can be used as candidate specific barcodes of Pulsatilla.However,their specific species discrimination needs to be further evaluated.