| Isatis indigotica Fort.is a biennial herbaceous plant of Isatis in the family Cruciferae,as the famous traditional Chinese medicine(TCM),the dried root and leaf of which are Isatidis Radix(Ban-lan-gen)and Isatidis Folium(Da-qing-ye)respectively.Isatidis Radix and Isatidis Folium with the effect of heat clearing,de-toxicating,cooling blood,relieving sore throat,anti-inflammation,attending fever and pharyngodynia,polydipsia,et al.I.indigotica mainly depend on cultivation and primarily distribute many provinces of China,such as Hebei,Anhui,Gansu,Heilongjiang,et al.In this study,all of thirty nine populations from fourteen provinces were collected and analyzed from morphology,molecular level and plant secondary metabolites level through numerical taxonomy,molecular biology and chemistry of TCM,and to discuss the genetic structure and variation,investigate the characteristics of genetic diversity of I.indigotica,and its relationship with the quality of medicinal materials.The results of this study will provide theory basis for the rational exploitation of I.indigotica.The main contents and results of this study are as follow:(1)Sixteen phenotypic traits of I.indigotica 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 leaf length,leaf width,leaf area,main root length,main root width,xylem were main factors for phenotypic diversity of I.indigotica.The thirty nine populations of I.indigotica were divided into 4 groups with morphological characteristics.(2)The Pollen grains of different populations in I.indigotica were observed and compared by means of scanning electron microscope.Pollen grain is long sphercity to super long sphercity with elliptical shape in equatorial plane,especially the polar front appears trifid circular and three monosulcate.Sulcus long almost reaching the ends of the grain.Reticulate exine ornamentation and grid mesh are generally pentagon or hexagon to irregular polygon.It is worth noting that the internal of grid mesh and its periphery distribute different density particular matter and cylindric columella respectively.The characters of I.indigotica populations were different in pollen size,pollen shape muri width,cellular size and cellular density.The thirty nine populations of I.indigotica could be divided into 4 groups according to the morphological characteristics of pollen.(3)Genetic diversity of I.indigotica populations was analyzed with IS SR molecular marker.At species level,polymorphism loci percentage(PPL)was 100.00%,the Nei's diversity index(He)was 0.4371 in average.At population level,PPL was 42,07%?71.03%,with an average of 59.68%.The populations of Xinjiang Hetian.Sichuan Qichuan and Hebei Wen'an have higher value about He and I than other areas and have abundant genetic diversity.Nei's genetic differentiation coefficient(Gst)was 0.4916,indicating a greater genetic variation within populations,and 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 similary.All of I.indigotica populations were divided into 5 groups according to their geographical distance relationship.(4)Genetic diversities and genetic structures of I.indigotica populations were analyzed with SRAP molecular marker.At species level,PPL was 98.76%,He was 0.3687 in average,Shannon information index(I)was 0.5320.At population level,PPL was 40.88%?70.35%,with an average of 57.55%,which show that the population of Gansu Minle was the highest one and Hebei Dingzhou present the stark contrast with the lowest.Furthermore,Gansu Minle,Sichuan Qingchuan and Xinjiang Hetian have the higher value of He and I than other populations,and have abundant genetic diversity.Nei's genetic differentiation coefficient(Gst)was 0.4687,indicating a greater genetic variation within populations,and there were also some differentiation among populations.The clustering analysis illustrates that the thirty nine populations were separated to 4 groups when the similarity factor was 0.735,then,the results from UPGMA and PCoA method showed that the analysis results were basically consistent with IS SR result,but had a little difference.(5)Ribosomal ITS2 sequences of different populations of I.indigotica were analyzed and compared.The length of ITS2 sequence was 191 bp,the content of G and C was 56.54%.The whole ITS2 area were found 1 simple mutation locis and haplotype diversity(Hd)was 0.689,nucleotide polymorphism(Pi)was 0.00803,the average number of nucleotide differences(K)was 1.533.Moreover,thirty nine populations genetic differentiation distance range from 0 to 0.0053,population of Hebei Dingzhou has higher level genetic differentiation.The phylogenetic system tree constructed by UPGMA method illustrated that the populations can be divided into 2 groups.In addition,ITS2 can distinguish I.indigotica from other plants of Isatis,it can be recommended that ITS2 is a suitable DNA barcode for identifying I.indigotica.(6)The chloroplast trnl-F sequences of different populations were analyzed.The length of trnl-F' sequence was 758 bp?760 bp,the content of G and C was range from 35.62%to 35.84%and average was 35.76%.The whole trnl-F area were found 3 simple mutation locis,all of them were single nucleotide polymorphic sites.Haplotype diversity(Hd)was 0.778,nucleotide polymorphism(Pi)was 0.06929,the average number of nucleotide differences(K)was 1.875.Thirty nine populations genetic differentiation distance range from 0 to 0.0013.The UPGMA tree show that the results were basically consistent with ITS2 data,but had a little difference.(7)To understand variation type among the different populations,this study tested the chloroplast rbcL sequences and analyzed the characteristics.It was showed that the length of rbcL sequences range from 553 bp to 555 bp,G and C content was 43.16%?43.60%and average was 43.28%.The rbcL sequences were found 4 mononucleotide mutation locus in total and genetic differentiation distance ranges from 0 to 0.0017.The UPGMA tree imply that thirty nine populations were divided into 2 groups.By comparing with ITS2 and trnL-F data,the results had slightly difference,but most tend to consistentcy.(8)The different populations and different parts in the same populations of I.indigotica are investigated and evaluated in quality according to some evaluation index for bioactive constituents,such as epigoitrin,indigo,indirubin,total alkaloids,polysaccharides,free amino acid,et al.The results show that the dominant chemical composition content of different populations have dramatically difference,the epigoitrin content ranges from 1.166 mg·g-1 to 3.912 mg·g-1,indigo content locates from 2.080 mg·g-1 to 8.630 mg·g-1,indirubin content range was 0.895 mg·g-1?2.751 mg·g-1.It was worth noting that the total alkaloids content of leaf in the same populations(1.027%?3.827%)was higher than root(0.089%?1.255%),however,the polysaccharide(3.784%?7.852%)and free amino acid content(1.494%?3.546%)in root were higher than the leaf.It can be observed that epigoitrin,indigo,indirubin,polysaccharide and free amino acid of leaf and root show rising trend with the latitude increasing.The thirty nine populations can be separated to 4 groups through clustering method.Germplasm selection can be carried out based on different requrements.It provided theoretical support for quality evaluation and development production of genuine Chinese medicinal.(9)The contents of 9 inorganic elements in different populations and different parts in the same population were determined by means of ICP-AES.The results showed that the contents of K and Ca were relatively higher than the contents of the other macroelements,and the content of Al was the highest in all of trace elements,the Fe and Zn rank only second to Al.The contents of each inorganic elements appeared significant difference in I.indigotica populations.It can be implied that the content of all the macroelements in leaf and Zn,Mn trace elements are all higher than in root,inorganic elements appear dramatically correlation.The principal component analysis showed that K,P,Mg,Fe,Al and Mn elements were the dominant factors.The thirty nine populations of I.indigotica were classified to 4 groups according to the inorganic elements contents. |
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