A Basic Study Of Genetic And Chemical Substances In Huanghua

Dao-di herb is a traditional Chinese medicine long-term clinical practice recognized as a good quality Chinese herbal medicine with the best quality and curative effect.It is a unique and comprehensive standard for appraising the quality of traditional Chinese medicines since ancient times.Clarifying the genuineness of the material base is an essential prerequisite for the sustainable use of Dao-di herbs.Scutellaria baicalensis Georgi(Lamiaceae)is the source of the well-known traditional Chinese medicine "HuangQin"(Radix Scutellariae),which has been extensively used in clinics to treat epidemic febrile disease and hyperactivity cough.Modern research in pharmacology and therapeutics has shown that S.baicalensis have been demonstrated in various disease conditions,including antitumor,anti-oxidant,anti-viral effects,anti-inflammatory,anti-bacterial,anticonvulsant,and hepato-protective.It is located on the 12 provinces north of the Qinling Mountains in China.It's main ecological environments include meadow grassland,coniferous and broad-leaved mixed forest,shrub and mountain grassland.The large-scale cultivation of S.baicalensis has begun since 1958.It is produced by Hebei Chengde and surrounding areas as genuine medicinal materials,as"Dao-di Huangqin".Huangqin is a perennial herb,with fleshy roots,insect-borne pollination,and seed reproduction.It's distribution,cultivation conditions,breeding systemand medicinal properties represent a large class of medicinal plants.It is a good example to study the genetic and chemical material basis research on the formation of Dao-di herbs.In this study,the genetic diversity and genetic structure of 28 wild populations were estimated using three polymorphic chloroplast fragments and ten pairs of SSR loci.A rapid,simple and sensitive method using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)technique combined with multivariate statistical analysis were employed to investigate whole metabolic profiles the differences in whole metabolic profiles and chemical composition in S.baicalensis from different regions.Correlative analysis was carried out to explore the genetic and chemical material basis of Huangqin genuiness.Then we can provid scientific suggestions about introducing,cultivation and breeding of S.baicalensis,which could lay a foundation for exploring the conservation and utilization of Huangqi germplasm resources.The main results are as follow:1.Chloroplast Genome Sequence of S.baicalensisWe developed valuable chloroplast molecular resources by comparing intraspecific and interspecific chloroplast genome.The total lengths of the sequences from the two S.baicalensis genotypes were determined to be 151,817 and 151,824 bp,with a circular quadripartite structure similarly to that of major angiosperm chloroplast genomes.A total of 114 genes were annotated,including 80 protein-coding genes,30 tRNA genes,and 4 rRNA genes.Comparative analyses of three Scutellaria chloroplast genomes revealed six variable regions(trnH-psbA,trnK-rpsl6,petN-psbM,trnT-trnL,petA-psbJ,and ycfl)that may be used as DNA barcodes.There were 25 single nucleotide polymorphisms(SNPs)and 29 indels between the two S.baicalensis genotypes.All indels occurred within non-coding regions.Phylogenetic analysis suggested that Scutellarioideae is a sister taxon to Lamioideae.These resources could be used to explore the variation present in Scutellaria populations and for further evolutionary,phylogenetic,barcoding and genetic engineering studies,in addition to effective exploration and conservation of S.baicalensis.2.PhylogeographyThe genetic diversity and genetic structure of 28 wild populations of S.baicalensis and 1 wild population of S.rehderiana were estimated using three polymorphic chloroplast fragments(ycf4-cemA,petA-psbJ and psbA-trnH).There was significant genetic differentiation between S.baicalensis Georgi and S.rehderiana,with no gene penetration and hybridization between species.The joint analysis of three cpDNA fragments of S.baicalensis revealed 29 mutation sites,forming 50 haplotypes,of which Hapl7,Hap20,Hap26,Hap27,Hap28,Hap32,Hap34,Hap35,Hap36,Hap37 and Hap43 can be used as a molecular geography marker for the identification of Dao-di Huangqin.There was a high haplotype diversity(Hd= 0.88)and nucleotide diversity(? = 1.58)within Huangqin.Dao-di Huangqin samples showed higher genetic diversity than non Dao-di Huangqin samples.In addition,the area of Dao-di Huangqin is very small in the entire distribution area,but it has concentrated 40 haplotypes,accounting for 80%of all haplotypes,so the Dao-di Huangqin is also a hub for the diversity of Huangqin.AMOVA analysis showed that the genetic differentiation mainly occurred among populations(75.84%)and the degree of genetic differentiation was higher(FST= 0.758).The gene flow between populations was lower(Nm = 0.1596).The analysis of the correlation between genetic distance and geographic distance showed a significant correlation(r = 0.33,P<0.01).The haplotype phylogenetic tree and geographic distribution map showed that the most primitive haplotype distribution area is Dao-di area.NJ clustering based on genetic distance and STRUCTURE clustering analysis based on Bayesian algorithm are support the division of 28 wild populations into three groups.The haplotypes expressed by the populations in Chengde and its surrounding area were Dao-di area(North China group).The haplotypes of the Qinling-Huai River(Shaanxi,Gansu,and Henan),Shandong and Shanxi are the northwest group,and the haplotypes in the northeastern provinces and Inner Mongolia are the northeastern group.Analysis of population dynamics showed that the number of effective populations in the Dao-di region was significantly greater than that in the non Dao-di region.There are two-way gene flow between the Dao-di region to the non Dao-di regions,but there is no gene flow within the non Dao-di regions.It is concluded that the Dao-di area of Huangqin is the origin and diversification center of S.baicalensis.3.Population GeneticsThe genetic diversity and genetic structure of 28 wild populations of S.baicalensis were ananlyzed using 10 pairs of SSR loci.High genetic diversity was detected in the Dao-di region(Ho = 0.568,He = 0.633,Na = 3.395,Ne = 3.395,I = 1.287).It is assumed that the Dao-di area may be the center of genetic diversity of S.baicalensis.The variance analysis of molecular variance revealed that the genetic variation was mainly found in the population of S.baicalensis(87.85%),the genetic differentiation among populations was smaller(FST ?0.1391),and the gene flow was larger(Nm = 1.5472),which was the opposite of the result of chloroplast fragments analysis.It indicates that for the genetic flow of Huangqin,the contribution of pollen flow is much greater than that of seed flow.The genetic distance and geographical distance of the population were significantly correlated(r = 0.61,P<0.01),but not significantly correlated with the altitude distance,indicating that the geographical factors of genetic differentiation among populations were mainly caused by spatial distance.The UPGMA clustering showed that the Dao-di area is scattered in the non Dao-di areas,which was consistent with that of cpDNA.The STRUCTURE clustering analysis further validates that two non Dao-di areas have evolved independently.The genotypes of the populations in the Chengde and surrounding areas are North China group,the genotypes of the populations of the Qinling-Huaihe River(Shaanxi,Gansu,and Henan),Shandong and Shanxi expressed northwest group and the populations of the northeastern provinces and Inner Mongolia are the northeastern group.It is assumed that the Dao-di area may be the origin of Huangqin.Combined with the cpDNA data,we further identified the Dao-di Huangqin,namely Chengde and the surrounding area,which may be the diversification center and origin center of Huangqin.The Yanshan Mountains in the north and the Taihang Mountains in the west form the natural barriers of Huangqin,which make it possible to become a refuge for plants in the northeast and northwest of the glacial period.The superior geography and climatic conditions and suitable ecological environment have formed the geoherbalism of Huangqin.11 kinds of unique haplotypes in the Dao-di area can be used as the molecular geographical indications for the identification of the Dao-di Huangqin,which is also the genetic material basis of the geoherbalism of Huangqin.4.MetabolomicsUPLC-Q-TOF-MS combined with multidimensional statistical analysis method was used for metabolomic analysis of 28 wild populations of S.baicalensis.29 ion peaks were detected,component identification was carried out according to the accurate m/z of primary mass spectrometry and the characteristics of secondary mass spectrometry,combined with the relevant literatures and databases.Principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA)and other multivariate statistical techniques were used to cluster the different metabolic profiles and chemical compositions,revealing the differences components of S.baicalensis from three different origins.In the three chemical-type,the populations in the Dao-di area is obviously different from the populations in the non Dao-di area,which is basically consistent with the genetic clustering grouping.Four different compounds were identified,namely Dihydroxy-dimethoxyflavone-7-glucuronide,Wogonin,Apigenin-7-diglucuronide and Trihydroxy-methoxyflavone glucuronide can be used as biomarkers.The contents of four different components in different producing areas have different degrees of difference.Among them,the content of Apigenin-7-diglucuronide and Trihydroxy-methoxyflavone glucuronide in the Dao-di area is significantly higher than that in other producing areas.They can be used as a chemical indicator of the Dao-di Huangqin,which is also the basis of geoherbalism chemical substances.Excellent geographical and climatic conditions and suitable ecological environment have resulted in the lineage sorting and the formation of a genetic differentiation model of modern Huangqin.This genetic differentiation model determines the geographical variation pattern of chemical phenotypes,and finally forms the geoherbalism of S.baicalensis.Conclusion:There is a certain correlation between the geographical changes of chemical composition and the genetic differentiation of chloroplast haplotypes and the genetic differences of nuclear genes,which are largely influenced by genetic factors.Although the native population is not an evolving significant unit,it has apparently specialized haplotypes and genotypes.The superior geographic environment of Dao-di district in the center of Huangqin distribution area preserves many specialized haplotypes and genotypes,which makes it possible to express high quality.Therefore,the formation of Huangqin geoherbalism should be the interaction between genetic and environmental.