Study On Genetic Diversity Of Germplasm In Tulipa Edulis (Miq.) Baker And Quality Evaluation On Its Materia Medica

Tulipa edulis(Miq.)Baker is a perennial herb in the family Liliaceae.The bulb has been used in traditional Chinese medicine(TCM)under the common name "Guangcigu",and has been used in the treatment of sore throat,scrofula,ulcer and postpartum blood stasis,et al.According to the records in Flora of China and our actual survey results,T.edulis distributes mainly in Anhui,Jiangsu,Zhejiang,Jiangxi,Hubei,Shandong,Henan,Shanxi and Liaoning provinces.Guangcigu is used more and more widely in clinical application with the development of its medicinal value,and market demands for Guangcigu also increased fast year by year.But the natural regeneration of T.edulis is very slow,and its wild resources become increasingly scarce.Therefore,it is very urgent to develop artificial cultivation techniques of T.edulis,and the studies on genetic diversity of germplasm in T.edulis are the foundation of effective protection and reasonable use of T.edulis.At presents,the studies about genetic diversity of germplasm in T.edulis is blank at home and abroad,and there is also the lack of comparison and analysis about phenotypic traits,genetic variation among different populations of T.edulis.In addition,the quality of TCM is determined by its genetic characteristics and growth environment,and mainly by genetic factors.Hence,it is particularly urgent to carry out the resources investigation and the studies on genetic diversity of T.edulis.In this study,all of twenty-eight germplasms of T.edulis,which were collected from Anhui,Jiangsu,Zhejiang,Jiangxi,Hubei,Shandong,Henan,Shanxi,Jiangxi and Liaoning provinces,were analyzed from morphology,palynology,DNA and metabolites level,and to discuss the characteristics of genetic diversity of T.edulis,and its relationship with the quality of medicinal materials.The results of this study will provide theory basis for the effective protection and rational exploitation of T.edulis.The main contents and results of this study are as follow:(1)Based on the field resources investigation of thirty wide spots in ten provinces,the investigation results showed that the resources of T.edulis distribute according to river basin,and mainly distribute in the Yangtze River,the Yellow River,Huaihe River and Bohai Rim,which are concentrated in the middle and lower reaches of Yangtze River and Huaihe River basin,such as central and northern of Jiangsu and Anhui provinces.Principal component analysis results showed that the geographic and climatic factors,which affect the distribution of T.edulis,were mainly annual active accumulated temperature,annual precipitation and annual sunshine hour.But the habitat types of T.edulis were rich,and it had a wide adaptability to temperature,light,humidity and soil types.Habitat types have a certain influence on reproductive system of T.edulis.(2)10 phenotypic traits of T.edulis from different populations were analyzed,and the results showed that there were highly significant differences with abundant genetic diversity.Principal component analysis results showed that the bulb diameter and bulb fresh weight were main factors for phenotypic diversity of T.edulis.(3)Pollen grains of 28 populations in T.edulis were observed and compared by means of scanning electron microscope.Pollen grain is monosulcate,inoperculate,boat-shaped,ellipsoid to long ellipsoid in polar view.Sulcus long,reaching the ends of the grain,shallow or broad.The characters of T.edulis populations were different in pollen size,pollen shape and pollen exine ornamentation.Main differences in exine ornamentation were ornamentation type,muri width,cellular shape,cellular diameter and cellular density.The pollens were divided into two class according to exine ornamentation,class ? was striate pollen,and class ? was reticulate pollen.According to muri width,class ? can also be divided into two categories,type ? a and type ? b.The muri of the type ? a<0.40 ?m,was formed by 1 or 2-5 shortliraes in different position to each other,and lumina is larger.The muri of the type ? b>0.40?m,was formed by 2-n shortliraes in different position to each other,the muri was more wider,and lumina is smaller.(4)Genetic diversity of T.edulis populations was analyzed by ISSR molecular marker.At species level,polymorphism loci percentage(PPL)was 94.80%,the Nei's diversity index(He)was 0.2648 in average.At population level,PPL was 12.80%?40.40%,with an average of 28.23%.Nei's genetic differentiation coefficient(Gst)was 0.533,indicating a greater genetic variation among populations,and there were also some differentiation within populations.The results from UPGMA and PCoA method showed that the all of T.edulis populations were divided into five groups according to their geographical distance relationship.The results from Mantel analysis showed that there were a certain correlation between genetic distances and geographic distances of different T.edulis populations.(5)Genetic diversities and genetic structures of T.edulis populations were analyzed by SRAP molecular marker.At species level,PPL was 100.00%,He was 0.3311 in average,Shannon information index(?)was 0.4873.At population level,PPL was 28.48%?57.44%,with an average of 43.62%,there was a higher PPL value in the populations of Xinxiang,and Xixian,Henan province.The higher He and ? values were found in the populations of Xinxiang,Henan province,Dalian,Liaoning province,Xixian,Henan province,indicating abundant genetic diversities.The value sizes of He and ? had a same change trends with PPL.Nei's genetic differentiation coefficient was 0.5024,indicating that there were a little difference between genetic variation within populations and among populations.The results from UPGMA and PCoA method showed that the analysis results were basically consistent with IS SR result,but had a little difference.(6)To understand variation type within species,we tested the ribosomal ITS2 base sequences and analyzed the characteristics of them.The effective length of ITS2 sequence was 474 bp,the content of G and C was 60.1%in average.A total of 18 mutation locis were found in ITS2 sequence,of which 14 single nucleotide polymorphic sites,4 parsimony informative polymorphic sites.Haplotype diversity(Hd)was 0.561,nucleotide polymorphisms(Pi)was 0.0042,the average number of nucleotide differences(K)was 1.974,indicating a certain degree of genetic variation.The results of phylogenetic analysis by UPGMA and PCoA showed that there wereat least three types of variation within T.edulis populations.Tongbai Mountains,Henan province,formed a monophyletic group branch,the other populationswere divided into north groups and south groups according to their geographical distance.(7)Chloroplast matK sequences of different populations of T.edulis were analyzed and compared.The results showed that the effective length of matK sequence was 886 bp,the content of G and C was 31.6%in average.A total of 36 variable sites were found in matK sequence,of which 22 single nucleotide variation sites,14 parsimony informative polymorphic sites.Hd was 0.997,Pi was 0.00651,K was 5.766.Compared with ITS2 sequences,matK sequence provided more abundant genetic informations.The results of phylogenetic analysis by UPGMA and PCoA showed that the result based on matK genetic distanceswas consistent with ITS2 result.It had obvious advantages by using matK combined with ITS2.(8)We selected matK,rbcL and psbA-trnH regions to examine the effective of these three regions as barcodes for T.edulis.The results showed that rbcL and psbA-trnH regions were very conservative.Together with other species from the order Liliaceae,the UPGMA tree of psbA-trnH and rbcL DNA sequences correctly placed T.edulis and its adulterants within the genus Tulipa,but T.edulis and its adulterants could not be distinguished from each other based on the sequence divergences.Whereas more abundant variation sits were found in matK sequences of Tulipa.Based on these SNPs in matK sequences,T.edulis and its adulterants can be differentiated.By combining these samples with other plants of the family Liliaceae,the matK based UPGMA tree could accurately identify T.edulis and its adulterants.Therefore,the matK region can be employed as the barcode for the authentication of T.edulis and its adulterants.(9)Extraction and determination method of polysaccharide in Guangcigu were established,and polysaccharide contents in Guangcigu from different origins were assayed.The results showed that polysaccharide contentsin Guanagcigu from different origins were from 8.1593 to 206.01 mg·g-1,and there was significant difference in polysaccharide contentsamong different samples.The highest polysaccharide content was found in the sample from Taishan,Shandong province,and the lowest content was found in the sample from Huzhou,Zhejiang province.Polysaccharide content in Guangcigu was correlated with latitude,temperature and lightfactors.From south to north in China,there was an increasing trend for polysaccharide content in Guangcigu.A new and rapid analytical method for the determination of polysaccharide in Guangcigu was developed by using near infrared spectroscopy combined with partial least squares regression method.The associated models of NIRS in calibration set and the corresponding content of polysaccharide was established by the chemometrics.The root mean squared error of cross-validation was 0.0031,and the coefficient of determination R2 reached 0.9994.The results showed the analyzed results were very satisfactory by this model,and this model can be applied for the rapid determination of large number of samples.(10)The contents of 26 inorganic elements in Guangcigu from different origins were determined by meansof ICP-AEC.The results showed that the contents of K,Ca and Mg were relatively higher than the contents of the other macroelements,and the content of Mn was the highest in all of trace elements.The contents of each inorganic elements appeared significant difference in Guangcigu from the different origins.Principal component analysis showed that K,Ca and Mg elements were the dominant factors which caused the difference among the samples from different origins.The contents of Pb,As,Cu,Cr and other heavy metals in all Guangcigu samples were very low,and they all conformed to the standards of Chinese Pharmacopeias.A new and rapid analytical method for the determination of inorganic elements in Guangcigu was developed by using near infrared spectroscopy combined with partial least squares regression method.The results showed that the quantitative models of inorganic elements in Guangcigu had some analysis ability,but the analysis effect was different for different elements,the effects of analysis for Pb,Ni,Sn and Fe were better,but were worse for the other elements.Therefore,we should continue to collect more samples to optimize the model.