Research On Polygonatum Germplasm Resources Based On Sugarome, Metabolome And Transcriptome

Polygonatum sibiricum Red is a genus of Liliaceae Polygonatum plant.Its perennial dried rhizome is considered a traditional Chinese medicine homologous with food.With mild properties and sweet taste,Polygonatum sibiricum Red has the effect of moistening lung and invigorating spleen,and nourishing Yin and tonifying kidney,which is widely used in the treatment of deficiency of spleen and stomach,pulmonary hemoptysis,diabetes,hyperlipidemia and so forth.In the 2015 edition of "Chinese Pharmacopoeia",three kinds of the original plants of the Chinese medicine are recorded:P.sibiricum,P.cyrtonema Hua,and P.kingianum Coll.et Hemsl.Studies have shown that the main medicinal ingredient of Polygonatum sibiricum Red is polysaccharide,which helps improve immunity,reduce blood sugar and lipid,anti-tumor,anti-bacteria and anti-virus.However,with the increasing application of polygonatum in the fields of medicine,food and other industries,the wild plants have been severely damaged and gradually exhausted.Currently,cases are not uncommon using other genera of polygonatum to replace P.sibiricum Red as medicine,such as P.punctatum,P.macropodium,P.filipes,P.cirrhifolium,and so on.To solve the contradiction between supply and demand,large-scale cultivation is the most effective and feasible method.The characteristics of P.sibiricum are the key factors in determining the yield and quality of the cultivated P.sibiricum.Primarily depending on asexual reproduction,continuous asexual reproduction of the current planted P.sibiricum gradually showing the reduced situation of resistance,yield and quality,leading to cultivate fine varieties has been very urgent.P.sibiricum Red is widely distributed,and under the comprehensive effects of fertilization and climate conditions at different regions,the germplasm vary greatly.It is of great practical and theoretical significance to systematically study different germplasm resources,establish scientific evaluation system of germplasm resources,select the excellent germplasm,and lay the foundation for the breeding of new variety.But there is still a lack of systematic research in this area.By screening the genes related to sugar synthesis pathway and the key enzyme genes,selecting the enzyme gene with SSR locus,and by constructing the DNA fingerprint of P.sibiricum based on SSR marker,On the basis of collecting and sorting on the germplasm resources of P.sibiricum,the phenotypic traits of different germplasms were screened out,and then the content and composition of the polysaccharides,transcriptome and metabolites were analyzed systematically.Ultimately,a complete set of methods for evaluating the quality of P.sibiricum was established and a database of core germplasm was constructed.Main research results:1.In our research,314 different kinds of germplasm resources from 19 groups in 11 provinces including Shaanxi,Gansu,and Henan were collected and sorted.According to the biological properties of P.sibiricum,taken into consideration their related traits,22 phenotypic trait indicators were chosen for statistical analysis,including leaf width(LW),leaf shape index(LSI),leaf round number(LRN),total pedigree length(TPL),total flower number(TNF),seed number(SN),root length(RL),rhizomes fresh weight(RFW)and so on.Seven characteristic indexes were found to be suitable for distinguishing germplasm differences,and they are RN(coefficient of variation 29.92%),LRN(22.54%),LSI(18.76%),LW(16.22%),TPL(14.74%),TNF(14.23%),SN(14.02%).The UPGMA cluster analysis was carried out according to the Euclidean distance of the phenotypic characters,and there were four categories of copolymerization,which showed a certain degree of regionality and discontinuity.According to the correlation analysis of phenotypic traits and ecological factors,we found that the longitude was very significantly positively correlated with pedicel length.The latitude and leaf width were very significantly positively correlated with current rhizome length.ATT(? 10 ? accumulated temperature/?)was very significantly positively correlated with total flower number,and positively correlated with root length,root number,root index,and current rhizome diameter.AR(annual rainfall),ARH(annual relative humidity),AMT(annual mean temperature),ATT(?10 ?accumulated temperature/?)were very significantly negatively correlated with current rhizome length.The wild distribution of P.sibiricum is consistent with the soil in the wet and watery slopes,and also provides a theoretical basis for the selection of sandy soil and ridge planting method for artificial cultivation of P.sibiricum.The results of correlation analysis showed that:when LW was wider,LSI would be smaller and CRL longer;The more the LRN,the heavier the RFW and the higher the PH;The longer the TPL,the heavier the RFW;The greater the TNF,the thicker the CRD;That is to say,the four phenotypic indicators of LW,LRN,TPL,and TNF are of great guiding significance to the selection of fine traits and the breeding of new varieties of P.sibiricum Red.2.According to the four phenotypic traits and the results of Q-type clustering,13 species of P.sibiricum were selected,and four kinds of P.cyrtonema and P.kingianum germplasms were used as contrast.The contents of polysaccharides in different germplasms were compared,from which the difference was found obvious.The polysaccharide content of P.sibiricum from Lueyang,Shaanxi Province(SXLY)was 13.33%,while the content of polysaccharide in Luoyang,Henan Province(HNLY)was 6.37%.The monosaccharide composition of crude polysaccharides was found the same in different germplasm resources,which were all composed of the four monosaccharides,including arabicose,mannose,glucose and galactose,but the molar ratio is different.The proportion of arabinose is the lowest,while the proportion of mannose is the highest,and the glucose and galactose are in the middle.When the proportion of arabinose was 1,mannose<18,glucose<3,and galactose<2.4,the polysaccharide content of the Polygonatum germplasm was higher;when the ratio of arabinose was 1,mannose>19.5,glucose>3.2,and galactose<2.6,the polysaccharide content of the polygonatum germplasm was lower.The three kinds of herbs listed in the"Chinese Pharmacopoeia",P.sibiricum,P.cyrtonema,and P.kingianum vary widely in monosaccharide composition.Through polysaccharide extraction,purification,and then further analysis the main physical properties of SXLY polygonatum germplasm.3.Taken into account the four key indicators of LW,LRN,TPL,and TNF,the weighted approach was adopted,Finally,the polygonatum germplasms from Guozhen at Lueyang,Shaanxi(SXLY-107-128),Fuping at Shaanxi(SXFP-129-142),Liuba at Shaanxi(SXLB-073-086),and Luoyang at Henan(HNLY-001-016)were selected for metabolic group and transcriptome study.Based on UPLC-Q-TOF/MS/MS technology,results showed that species of polygonatum germplasms(SXFP/SXLY/HNLY)are less differentiated,while the differences on the secondary metabolites of SXFP/SCMS/YNLC were significant.The potential markers were screened by partial least squares-discriminant analysis(PLS-DA)and s-plot analysis.Interspecies differences were greater than intra-species differences.Twenty-five landmark differential metabolites were identified by secondary mass spectrometry.Compound KEGG annotation analysis results show that the four metabolic pathways were significantly different,and they are starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism,galactose metabolism,and beta-Alanine metabolism.Thermal image analysis,box-type diagram of metabolites and Z-score analysis showed that,the signature difference metabolites that can distinguish different species of polygonatum and their germplasms are Adenosine,Sucrose and Pyroglutamic acid.This thesis provides a new direction for the differentiation of the polygonatum germplasms and related metabolic pathways.4.The two germplasms(SXLY and HNLY),which had the largest difference in polysaccharide content,were transcriptome sequenced,and 13,839 unigenes were annotated to 128 metabolic pathways.Among them,1,860 unigenes were associated with carbohydrate metabolism,and 20 genes(138 unigenes)were involved in the biosynthesis of polysaccharides from P.sibiricum.There were eight key enzyme genes(88 unigenes)that had significant differences between the two germplasms,and they were sacA,GALE,UGDH,UXE,RHM,scrK,GMPP,and HK.Through the GeNorm,NormFinder,BestKeeper software comprehensive analysis,we found that the most suitable internal reference gene of polygonatum is TUB.qRT-PCR was used to detect the expression levels of the above eight key enzyme genes in different germplasm resources.It was found that the polysaccharide content was positively correlated with the expression patterns of sacA,scrK,GALE,GMPP and UGDH,but negatively correlated with HK expression.This indicated that the expression of these genes can also be used to distinguish between different P.sibiricum germplasms.Through comprehensive analysis on the result of the composition and content of sugar,it showed that the key enzyme genes in the synthesis pathway of P.sibiricum were sacA,HK and GMPP,which could help understand the biosynthesis of polysaccharides from the molecular level,and also laid a solid foundation for the molecular identification of germplasm difference.5.Based on the sequencing analysis of P.sibiricum transcriptome,the enzyme gene with SSR locus in the synthesis pathway of P.sibiricum was screened and the primers were developed.Twelve pairs of core primers were used to construct DNA fingerprints of Polygonatum in 32 kinds,which include not only materials of different Polygonatum germplasms,but also of P.cyrtonema and P.kingianum.It is found that the fingerprints of each region are unique,and the clustering and PCA analysis could accurately reflect the genetic relationship of different varieties.The smaller the difference in polysaccharide content,the closer the clustering relationship.Conversely,the greater the difference in polysaccharide content,the further the clustering relationship.And all the germplasms of P.sibiricum,P.cyrtonema,and P.kingianum were respectively clustered together.Finally,the fingerprint QR coding was formed containing the name,type,botanical classification,germplasm source,climate situation and fingerprint code.The results of the study help improve to a large degree the breeding of fine varieties and excellent germplasm identification.To sum up,based on the germplasm resources,this thesis set polysaccharide research as the main line,and differential metabolic research as the auxiliary line,and it also established a whole set of scientific methods for evaluating the quality of P.sibiricum germplasms.