Researches On Population Genetics For Wild Populations Of Fritillaria Cirrhosa D.don And Its Related Species,as Well As Quality Evaluation Of The Medicines

Objective:Infrared spectroscopy,HPLC and AFLP molecular markers were used for species identification,quality evaluation and population genetics studies of F.cirrhosa and its closely related species.Through research and compare the differences in morphology and content between them to explore their complex genetic relationships and evolutionary history,providing reference for the conservation and clinical research of wild resources.In addition,this paper discussed phylogenetic position of Hemiphragma heterophyllum Wall.in the Scrophulariaceae and its relationship with other taxa based on the complete chloroplast genome.Methods:（1）Infrared spectral information was collected from 28 batches bulbus of F.cirrhosa and its closely related species.The spectral data were processed by automatic baseline correction,automatic smoothing,vertical coordinate normalization and second derivative.The infrared fingerprint was constructed and analyzed by principal components analysis（PCA）and hierarchical cluster analysis（HCA）.（2）HPLC method was used to establish the fingerprints of 26 batches Fritillariae Cirrhosae Bulbus from different origins and producing areas.The content of cytosine,uracil,cytidine,hypoxanthine,guanosine,thymidine,adenine,inosine,uridine,thymidine,adenosine and 2’-deoxyadenosine was determined and evaluated by PCA and HCA in Fritillariae Cirrhosae Bulbus.（3）The AFLP markers were adopted to study genetic diversity,population structure and evolutionary history of 30 populations（561 individuals）for eight F.cirrhosa and its closely related species.In addition,this paper also supplementary reported structure and characteristics of the complete chloroplast genome of H.heterophyllum,and discussed phylogenetic position of this species in the Scrophulariaceae and its relationship with other taxa.Results:（1）Fast identification of F.cirrhosa D.Don and its closely related species based on infrared spectroscopyThe results showed that main absorption regions of infrared spectra of the five F.cirrhosa and its closely related species were 700～400 cm^-1;950～700 cm^-1,1200～1000cm^-1,1500～1200 cm^-1,1800～1500 cm^-1 and 2800～3500 cm^-1.The second-derivative spectra in 3000～2000 cm^-1 and fingerprint region of 1800～500 cm^-1 had a certain difference,with obvious differences in peak potential,peak shape and intensity.HCA and PCA analysis results showed that all samples were clustered into two major categories.Among them,F.przewalskii Maxim.,F.crassicaulis S.C.Chen and F.sichuanica S.C.Chen could be distinguished significantly,but F.cirrhosa and F.unibracteata couldn’t be separated,which indicated that original plants of the Bulbus Fritillariae cirrhosae were relatively closer in relationship.（2）HPLC fingerprint and quantitative analysis of 12 nucleoside components for Fritillariae Cirrhosae BulbusA total of 15 common peaks were identified by HPLC fingerprint of Fritillariae Cirrhosae Bulbus,and nine common peaks and four non-common peaks were identified using the reference substance.The similarity of 26 batches of samples ranges from0.656 to 0.954.The similarity of medicinal materials from different areas with same origins was basically high,only F.unibracteata（S6,S7,S8）,F.cirrhosa（S10）,and F.unibracteata var.wabuensis（S25,S26）were relatively low.Content determination results showed there were some differences in the contents of 12 nucleosides and bases in Fritillariae Cirrhosae Bulbus from different places.Among them,the main nucleoside components were guanosine,uridine and adenosine,and the three nucleosides content were relatively higher in F.delavayi（S21）,F.taipaiensi（S22,S23,S24）,F.unibracteata var.wabuensis（S25,S26）.The results of HCA and PCA analysis were basically the same.The 26 batches Fritillariae Cirrhosae Bulbus were roughly grouped into four categories,and the medicinal materials from three sources with higher contents were gathered into one branch.（3）Population genetics researches of F.cirrhosa and its closely related species based on AFLP markerThe results showed that F.cirrhosa had the highest genetic diversity at the species level(H_SP=0.2960);whereas,F.dajinensis showed the lowest value(H_SP=0.1785).Moreover,at population level,mean value of genetic diversity was the highest in F.unibracteata(H_POP=0.2609),but the value was also lowest in F.dajinensis(H_POP=0.1785).In F.cirrhosa,HLS,DDS and DQ populations showed high genetic diversity,but YD and QJ possessed low variation.Furthermore,genetic differentiation among populations were relatively low(G_ST=0.0622～0.1561)which was similar to AMOVA analysis that only 19.15%were existed between populations.Finally,the 30populations were roughly divided into six clusters in UPGMA dendrogarm,of which F.cirrhosa was relatively complicated and composed of multiple lineages.STRUCTURE and PCo A analysis also supported the complex relationships among species and evolutionary lineages.The present study revealed that widely distributed species generally possessed higher genetic diversity than the narrowly distributed.In F.cirrhosa,populations in central Hengduan Mts.probably had higher protected value because of much richer genetic diversity than that in the margins of distributions.（4）Phylogenetic researches of H.heterophyllum Wall.based on complete chloroplast genomeThe results showed that genome sequences of two individuals for H.heterophyllum in this study were highly similar except slight differences on sequence lengths（152,707bp and 152,700 bp,respectively）.The complete chloroplast genome of H.heterophyllum was a typical circular quadripartite structure.A total of 113 genes were annotated,including 79 protein-coding genes,29 t RNA genes,four r RNA genes,and one pseudogene.Among the annotated genes,16 genes contained one intron,whereas another two genes（ycf3 and clp P）possessed two gene introns.Total GC content was38.1%in the chloroplast genome.Moreover,a total of 63 simple sequence repeats（SSRs）with five types were detected.Phylogenetic analysis（neighbor-joining tree,NJ tree）revealed that Hemiphragma was closely related to Veronica L.and Veronicastrum Heist.ex Farbic.Conclusion:（1）Compared with traditional character identification,infrared spectroscopy identification method had more advantages.This method could distinguish part of original medicinal materials,but its identification ability was limited,which could provide a reference for identification of F.cirrhosa and its related species.（2）The results of fingerprint and content determination indicated that there were some differences in contents of 12 nucleosides and bases in Fritillariae Cirrhosae Bulbus from different places,the main components were guanosine,uridine and adenosine.Possibly due to the influence of factors such as growth years and environment,the regularities between populations and species in the region were poor.The method was simple,accurate,reliable and reproducible,which can provide reference for the quality evaluation of Fritilariae Cirrhosae Bulbus.（3）The results of population genetics research indicated that there were extremely complicated genetic structure and evolutionary relationship between F.cirrhosa and its closely related species.The concept of“F.cirrhosa complex”,proposed by Luo et al.,did not get support from genetics and should be further revised.Furthermore,F.cirrhosa was a typical species with multiple origins and more researches should be carried out to provide more reasonable treatments in taxonomy.This method would provide new cognizing on evolution of F.cirrhosa and its closely related species,as well as was of great significance for conservation and sustainable utilization.（4）The complete chloroplast genome study was accurately revealed phylogenetic location of H.heterophyllum in Scrophulariaceae,which would facilitate further phylogenetic research on related species or genera of this family.