| Objective: To study the phenotypic characters,microscopic structure,physicochemical properties and DNA barcode fingerprints of 22 golden camellia species,to identify the differences in plant morphology,crude drug characters,chemical composition and DNA barcode,and to establish an interspecific classification and identification method;and analyze the genetic diversity and genetic relationship to provide reference for the accurate identification,quality control and resource utilization of all these species of golden camellia.Methods:(1)Phenotypic characters and microscopic identification methods were used to observe the original plant morphology and tissue structure of 22 golden camellia species,and to compare and analyze the differences in their appearance morphology,component types and tissue structure.(2)The common pattern of UPLC fingerprints of 22 golden camellia species was established by ultra high liquid chromatography(UPLC)combined with the similarity evaluation system of traditional Chinese medicine fingerprints,and their similarity was evaluated.Principal component analysis and orthogonal partial least squares discriminant analysis model in SIMCA-P14.1 software were used to analyze the principal components of the UPLC fingerprints to determine the differences in chemical composition of 22 golden camellia species.(3)General primers of ribosomal DNA second internal transcription spacer(ITS2),RNA transcriptor type II intron cleavage enzyme gene(mat K),chloroplast ribulose-1,5-diphosphate carboxylase large subunit(rbc L),chloroplast non-coding region(trn L-trn F,psb A-trn H)sequences were used for amplification by polymerase chain reaction(PCR),detection by electrophoresis and bidirectional sequencing.MEGA7.0 software was used to analyze the sequence variation,the cluster tree was constructed by using the neighbor-joining(NJ)method,and the secondary structure of sequences was predicted by RNAFOLD.The DNA information differences of 22 golden camellia species were analyzed,and the method of DNA barcode identification was established.(4)DNA restriction endonuclease Sacll was used to cut the sequences amplified with the primer rbc L,and the size differences of enzyme cut fragments were analyzed.A analytic method for polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)was developed.Results:(1)Results of phenotypic traits There were some differences in leaf type,leaf surface characteristics and flower organs of 22 golden camellia species.They had similar leaf shape,oval or long oval,and their size difference was obvious.Among them,C.chrysantha(Hu)Tuyama var.macrophylla S.L.Mo et S.Z.Huang was macrophyllous,with mature leaves15.5 ～ 27 cm in length and 6.8 ～ 19.5 cm wide,significantly larger than those of other species(4.6 ～ 21.5 cm long and 2 ～ 10 cm wide on average).The branches,leaves and flower organs of C.Pubipetala Y.Wan et S.Z.Huang were densely villose,unique to this species.The vein of C.impressinervis Chang et S.Y.Liang was deeply depressed on the upper surface,with numerous veinlets.C.nitidissima Chi showed uniquely 3 small brown spots on the top of the three outer petals.(2)Results of microstructure analysis Twenty-two kinds of Golden camellia species were studied.The results showed that the microscopic characteristics of other Golden camellia species were not much different except for the obvious characteristics of Camellia Pubipetala Y.Wan et S.Z.Huang and Camellia euphlebia Merr.et Sealy.There were significant differences in the structure of root,stem and leaf of 22 golden camellia species.Xylem vessels in the transverse section of the root of C.chrysantha were large and low in quantity.There were lignified cells in the center of xylem in the cross section of root of C.fusuien achrysantha Chang et S.Y.Liang.According to the number of stone cells in the stem cross section cortex and phloem,golden camellia species fell into two groups.The cortex and phloem in the stem cross section of C.nitidissima,C.fusuien,C.chrysantha,C.euphlebia and C.enchengensis had 2～3 layers of stone cells scattered in a circle,forming a ring of stone cells,while other golden camellia species stem cross section cortex and phloem displayed few or very few stone cells.Golden camellia species could be divided into three categories according to the layers of palisade tissue arranged in the transverse section of the leaves.The foliar palisade tissue of C.chrysanthoides was arranged into one layer in the leaves,that of C.enchengensis arranged into three layers,and that of other Golden camellia species arranged into two layers.Non-glandular hairs were present on the upper and lower epidermis of the transverse section of C.Pubipetala,which can be used to distinguish this species from others.(3)Results of chemical composition analysis There were significant differences in the UPLC fingerprints of 22 golden camellia species,chemical component types differing much with each species having its own unique peak(s),and much difference existing the peak area of common peak(s).Based on the peak area of chemical composition,9 principal components were obtained from 22 golden camellia species by principal component analysis,and the cumulative variance contribution rate was 76.70%.(4)DNA barcoding results DNA samples were extracted successfully from all species.Except with the ITS2 primer,amplification was successful using all other primers and all sequencing success rates reached 100%.The secondary structure of rbc L,mat K,psb A-trn H and trn L-trn F sequences of 22 golden camellia species were different in branch length,angle between branches as well as size,number and location of stem rings,based on which the 22 species could be distinguished from each other.However,phylogenetic trees based on single gene sequences of mat K,rbc L,trn L-trn F and psb A-trn H showed that 22 golden camellia species,C.oleifera and C.japonica had chaotic genetic relationships,although some species of golden camellia were more closely related to the latter two species.(5)Results of PCR-RFLP study Sequences amplified using the rbc L primers of C.fusuien achrysantha Chang et S.Y.Liang and C.fusuien achrysantha Chang et S.Y.Liang could not be digested by Sacll restriction endonuclide,and only 1 band appeared at 500 ～ 750 bp after enzyme digestion.After being digested by Sacll restriction enzyme,sequences amplified using the rbc L primers of the remaining 20 golden camellia species showed two bands between 100 and 250 bp and between 250 and 500 bp.Camellia fusuien achrysantha Chang et S.Y.Liang and Camellia fusuien achrysantha Chang et S.Y.Liang could be distinguished from other 20 golden camellia species by this method.Conclusion: Using methods of character identification,microscopic identification,physical and chemical identification and DNA molecular identification,alone or combined,22 golden camellia species can be accurately identified and classified.Our results may provide scientific data for the evaluation,development and utilization of golden camellia species germplasm resources and the improvement of quality standards. |
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