Microscopic And Molecular Identification Methods Of Asarum Heterotropoides Fr.Schmidt Var.mandshuricum In Germplasm Resources

A.heterotropoides Fr.Schmidt var.mandshuricum, A.sieboldii Miq f.seoulense and A.sieboldii Miq were all contained in 2015 edition of "Chinese Pharmacopoeia", A.heterotropoides Fr.Schmidt var.mandshuricum has the best quality, it has become the mainstream market varieties. To improve the clinical medicine security and controllable of Asarum, strengthen the production safety and quality standards of traditional Chinese medicine, promote accurate use the traditional Chinese medicine, identification of germplasm resources of A.heterotropoides Fr.Schmidt var.mandshuricum and its closely related plants were studied by comparative microscopic structure, biochemistry and DNA identification bar code identification methods. The following results:1. Leaf anatomical structures of A.heterotropoides Fr.Schmidt var.mandshuricum and its closely related plant were studied by means of paraffin method and Colorless nail polish imprint method. The results showed that the leaf microscopic structures of A.heterotropoides Fr.Schmidt var.mandshuricum and its closely related plants were in line with structure characteristics of the shade plants, but in the epidermal cell size and arrangement, palisade tissue, mesophyll cell morphology and arrangement, and the leaf stoma density existed difference. Leaf palisade tissues of A.heterotropoides Fr.Schmidt var.mandshuricum were composed of a layer of columnar cells, leaf palisade tissues of A.sieboldii Miq f.seoulense and A.sieboldii Miq were composed of two layers of columnar cells, leaf palisade tissues of A.caudigerum Hance var.caudigerum and A.caudigerum Hance var.cardiophyllum were composed of a layer of columnar cells, but the cells were small and arranged loosely. A.splendens and A.delavayi and A.pulchellum were no significant differentiation of palisade tissue. differences in palisade tissue, can be used as identifying characteristics of the A.heterotropoides Fr.Schmidt var.mandshuricum, also anatomically confirmed A.sieboldii Miq f.seoulense and A.sieboldii Miq between a close phylogenetic relationship. Chloroplasts in mesophyll cells of A.heterotropoides Fr.Schmidt var.mandshuricum, A.sieboldii Miq f.seoulense and A.sieboldii Miq were small and dense, chloroplasts in mesophyll cells of A.splendens and A.pulchellum were larger. chloroplasts in mesophyll cells of flower leaf A.caudigerum Hance var.cardiophyllum mainly distributed in upper mesophyll cells, chloroplast differences in mesophyll cells can be classification and identification reference for Asarum L.2. Seed protein electrophoresis spectrum showed that A. heterotropoides Fr. Schmidt var.mandshuricum and A.sieboldii Miq f.seoulense had 5 characteristic spectrum bands(A5, A6, B1, B2, B3), A.heterotropoides Fr.Schmidt var.mandshuricum had 5 characteristic spectral bands(A5, A6, B1, B2, B3), and 5 specific spectrum bands(A2, A3, A4, C1, C2); A.sieboldii Miq f.seoulense had 6 characteristic spectral bands(A4, A5, A6, B1, B2, B3),and 4 specific spectrum bands(A1, A3, C1, C2), but did not find the seed protein interspecific specificity band. Therefore, the use of seed protein electrophoresis can’t achieve the identification of A.heterotropoides Fr.Schmidt var.mandshuricum and A.sieboldii Miq f.seoulense seeds.3. The nuclear gene(ITS, ITS1,and ITS2) and chloroplast genetic(matK, rbcL,rpoC1, trnL-F, and psbA-trnH) were selected as candidate DNA barcode, to evaruate the effectiveness and identification of different candidate DNA barcode in Asarum L. The results showed that in the eight candidate DNA barcode, the nuclear gene ITS1 had the highest interspecific genetic variation and had relatively low intraspecific genetic variation, which had high identification rate(88.75%). Therefore, the nuclear gene ITS1 was suitable for Asarum L. identification. The nuclear gene ITS, ITS2 and chloroplast genetic trn L-F had large interspecific genetic variation. There was no significant differences in the interspecific and interspecific of the matK, rbcL, rpoC1 in the chloroplast genome, and psbA-trnH had difficulty in PCR-amplified. Therefore, nuclear gene ITS, ITS2, trnL-F and chloroplast genetic rpoC1 were not suitable for Asarum L.plant identification. Based on the above research, fast identification of Asarum L. plant can be realized combined with the genebank ITS1 sequences.