Study On Quality Evaluation Of Cortex Moutan Based On SSR Marker And Component Analysis

PurposeThe purpose of this study is to develop the SSR molecular markers of Cortex Moutan based on transcriptome sequencing data,and to analyze the genetic diversity of different populations of P.suffruticosa,and distinguish Cortex Moutan from different sources.A quantitative analysis method was established to evaluate the chemical qualities of six active components in Cortex Moutan.Methods and Results1.Data analysis of Cortex Moutan transcriptome sequencing The transcriptome sequences of two Cortex Moutan samples in chongqing were obtained by Illumina high-throughput sequencing,a total of 84,965 Unigene were screened based on transcriptome sequencing.MISA software was used to analyze Unigene,identify SSR types and analyze SSR site characteristics.Then 6,235 SSR were identified from 16,542Unigene longer than 1,000 bp units,mononucleotides were the most abundant SSR with a proportion of 67.31%,followed by 20.03%dinucleotides and 11.73%trinucleotides,tetranucleotide,pentanucleotide and hexanucleotide repeat types accounted for 0.93%in total.2.Development of SSR molecular markers of Cortex Moutan SSR molecular markers of Cortex Moutan were developed based on transcriptome sequencing data.Primers were selected from 6,235 SSR sites according to certain standards,and 132pairs were randomly selected after screening.Using Cortex Moutan samples from Chongqing as the material for experiment,83 pairs of primers could be effectively amplified,with an amplification efficiency was 62.88%.Among them,10 pairs of primers（P1-P10）were polymorphic primers.10 pairs of polymorphic primers were amplified in a total of 50 material genomic DNA from 5 P.suffruticosa populations to detect the genetic polymorphism of SSR primers.A total of 28 alleles were detected.The number of observed alleles（Na）range from 2 to 4;observed heterozygosity（Ho）range from 0.0816 to 0.6800;expected heterozygosity（He）range from 0.3232 to0.6455;Shannon’s information index（I）range from 0.5004 to 1.0944;polymorphism information content（PIC）range from 0.2688 to 0.5711.Primers P8 and P10 were highly polymorphic loci（PIC>0.5）,and the others were all moderate polymorphic loci（0.25<PIC<0.5）.3.Genetic diversity analysis of Cortex Moutan According to the amplification results of 10 pairs of polymorphic SSR primers,genetic diversity and genetic relationship of 5 P.suffruticosa populations were analyzed,and UPGMA method was used to construct a phylogenetic tree based on Nei’s genetic distance of 50 samples.For this 5 populations（“Taipinghong”simple flower,multiple flower and“Fengdan”from Chongqing,“Fengdan”from Anhui,ornamental cultivars of P.suffruticosa from Henan）,The Nei’s expected heterozygosity（H）range from 0.2580 to 0.3993,Shannon’s information index（I）range from 0.3639 to 0.6587.“Fengdan”from Anhui had the highest level of genetic diversity（H=0.3993,I=0.6587）,“Taipinghong”multiple flower from Chongqing had the lowest level of genetic diversity（H=0.2580,I=0.3639）.The genetic identity（GI）among the 5 populations ranged from 0.5363 to 0.9342,genetic distance（GD）varied from 0.0681 to 0.6231.The lowest genetic identity（GI=0.5363）and the farthest genetic distance（GD=0.6231）were observed between“Taipinghong”multiple flower from Chongqing and ornamental cultivars of P.suffruticosa from Henan,indicating that the two populations had the farthest genetic relationship.While the greatest genetic identity（GI=0.9342）and the shortest genetic distance（GD=0.0681）were observed between“Fengdan”from Chongqing and“Fengdan”from Anhui,indicating that the two populations had the closest genetic relationship.The 50 samples could be completely divided into three clusters based on genetic distance.All individuals of“Taipinghong”from Chongqing with simple flower type and multiple flower type first formed a small branch respectively and then gathered together as a big branch;All individuals of“Fengdan”from Chongqing and“Fengdan”from Anhui formed a branch;All ornamental cultivars individuals from Henan formed a branch.The results of cluster analysis show that the same species of P.suffruticosa have great genetic identity without being affected by geographical origins.The results could distinguish the medicinal and ornamental P.suffruticosa from each other.4.Determination of six components in Cortex Moutan The contents of gallic acid,oxypaeoniflorin,paeonolide,paeoniflorin,benzoylpaeoniflorin and paeonol in Cortex Moutan were determined simultaneously by HPLC.Cluster analysis of 50samples was carried out by using SPSS 20.0 software based on the content of six components.Samples were separated on an Ecosil C₁₈（250 mm×4.6 mm,5μM）.The linear gradient conditions were solvent A of 0.2%formic acid in deionized water and solvent B of acetonitrile using a gradient program of 0-3 min,10%-11.8%B;3-5 min,11.8%-12%B;5-7 min,12%-12.1%B;7-9 min,12.1%-12.2%B;9-10 min,12.2%-12.3%B;10-15 min,12.3%-13%B;15-18 min,13%-15%B;18-21 min,15%-15.8%B;21-26min,15.8%-74%B;26-30 min,74%-90%B;30-35 min,90%-90%B.Elution was performed with a solvent at the flow rate of 1.0 mL/min,and the injection volume was10μL for analysis.Ultraviolet detector was set at 274 nm and 230 nm for acquiring chromatograms of paeoniflorin,benzoylpaeoniflorin and gallic acid,oxypaeoniflorin,paeonolide,paeonol,respectively.The method validation showed that chromatographic conditions,linearity,precisions,stability,repeatability and recovery were well established.The HPLC conditions were suitable for the determination of six components in Cortex Moutan.The content determination results showed that all fifty samples meet the requirements of the 2015^th edition of Chinese Pharmacopoeia.Paeoniflorin and paeonol were the main components,the accumulations of two ingredients in“Taipinghong”simple flower from Chongqing were more than that in multiple flower type;the content of paeonol in“Fengdan”from the two geographical origin had little difference,the content of paeoniflorin in“Fengdan”from Chongqing was significantly higher than that from Anhui;the chemical quality of ornamental cultivars from Henan are instable and the contents of 6 components vary widely.The result of cluster analysis showed that the 50 Cortex Moutan samples were divided into two branches,The most samples sourced of“Taipinghong”from Chongqing included in one branch;“Fengdan”from Chongqing and and Anhui included in one branch without being affected by geographical origins;While ornamental cultivars samples from Henan were separated into two clusters.Conclusions1.Abundant transcriptome data of Cortex Moutan were obtained by high throughput sequencing,with well sequencing results.The assembly integrity of Unigene was high,and abundant SSR identified from Unigene.Transcriptome sequencing is one of the effective methods for the development of SSR molecular markers.2.The 10 pairs of SSR molecular markers were developed and screened in this study have well polymorphism,and cluster analysis based on genetic distance can clearly distinguish the two varieties of medicinal P.suffruticosa from ornamental cultivars.3.The varieties and geographical origins of Cortex Moutan are important factors affecting the content of chemical components.In summary,The SSR molecular markers combined with the multi-component quantitative analysis could be used as a effective method for quality evaluation of Cortex Moutan.