| Epimedium pubescens,an important plant species recorded in Chinese Pharmacopoeia2020 edition,is one of the main sources of Herba Epimedii(Epimedium),it is also a species that has been domesticated and cultivated on a large scale in China.Herba Epimedii,a kidney tonic herb used in traditional Chinese medicine,has been verified to have activities such as antiosteoporosis,enhancement of immunity,anti-tumor and neuroprotection by pharmacological studies.It is well-known that 8-prenylated flavonol and its glycosides are the main bioactive components,among which icariin-related flavonol glycosides are used as key components in quality control.Therefore,to determine the genetic basis affecting the chemical constitution of flavonoids and the content of main bioactive components in E.pubescens is crucial for cultivation of new varieties and quality improvement.Solving the above-mentioned problems can play a huge role in new varieties cultivation and quality promotion.However,only a few structural or regulatory genes have been identified in the biosynthetic pathway so far.In this study,we aim at understanding the composition and content characteristics of icariin-related flavonoids in the wild and cultivated populations of E.pubescens,determining the candidate population for breeding and re-sequencing.The selected populations can be further used for breeding utilization and candidate genes mining.1)Population selection of E.pubescens based on the difference of icariin-related flavonoids.A total of 39 populations were collected from all over China.UPLC/PDA-QTOF/MS,IGS sequence and geographic distribution were used to select populations with genetic characteristics,especially different composition and content of icariin-related flavonoids.Firstly,qualitative and quantitative research were carried out by conducting UPLCPDA-Q-TOF/MS and PCA analysis.As a result,a total of 41 kinds of icariin-related flavonoids were identified,and the flavonoid composition of icariin-related flavonoids were similar among different populations.Ten components with high content,accounting for 50～85% of icariinrelated flavonoids,were identified as the main components to conduct relative quantification,including Epimedin A1,Epimedin A,Epimedin B,Epimedin C,Icariin,3"-carbonyl-2"-β-Lquinovosyl-icariin,Sagittatoside A,Sagittatoside B,2"-O-rhamnopyranosyl icariside II and Icariside II.According to the relative content of different components in the above 10 icariinrelated flavonoids and the relative content ratios of Icariin and Epimedin C,39 populations were divided into 6 groups,and 27 populations were initial selected based on the fingerprint characteristics and geographical distribution.Furthermore,IGS sequences of different populations were determined,and 26 populations with different genetic characteristics were finally selected,which can lay foundation for candidate gene mining using re-sequencing technology.2)Population selection of E.pubescens based on the variation of icariin-related flavonoids content.HPLC was used to analyze the variation of total ABCI content(quality index of Herba Epimedii in Chinese Pharmacopoeia 2020 edition)in 37 populations of E.pubescens.Firstly,6individuals were randomly selected from each population for total ABCI measurement,and the results revealed that the coefficient of variation ranged from 8.08 to 56.35%.Furthermore,a secondary verification was applied for the selected candidate populations,featured with high or low content but with lowest variation in icariin-related flavonoids content.Finally,2candidate populations with small content variation were selected,which not only can be used as the backbone population of breeding,but also for candidate gene mining of icariin-related flavonoids and marker-assisted breeding through self-cross purification.3)Candidate genes mining for genes related to icariin-related flavonoids content.Firstly,HPLC method was applied to determine the total ABCI content of 500 samples from E.pubescens,named Guitong NO.1.Three samples were separately screened out as the target materials,which were with the highest and lowest content.Cluster analysis based on gene expression level from RNA-seq combined with variance analysis of icariin-related flavonoids content showed significant difference between high and low content groups.Among the 2,004 differentially expressed genes obtained,1,318 genes were significantly up-regulated in the high-content group compared with the low-content group,13 key structural genes of icariinrelated flavonoid synthesis pathway,35 downstream modification structural genes that may be involved in icariin flavonoid synthesis and 14 transcription factors were identified.The screened candidate genes can be used for gene function analysis,as well as for searching for regulatory factors affecting the content of icariin-related flavonoids.In this study,a comprehensive method combining UPLC-PDA-Q-TOF/MS,PCA,IGS sequence,geographic distribution and HPLC were used to reveal the variation characteristics of icariin-related flavonoids in wild and cultivated populations of E.pubescens.A total of 62 candidate genes were identified from individuals with different contents of icariin-related flavonoids,which provided an important basis for further analysis of icariin-related flavonoid content and laid a foundation for marker-assisted breeding for E.pubescens. |
PDF Full Text Request