| Schizonepetae Herba and Bupleuri Radix, as bulk Chinese herbs, are widely planted in China and enjoy a long history of medicinal application. Foundamentally, the germplasm quality and safety of herbs directly affect the quality and clinical efficacy of their products. In the past, the germplasm of Schizonepetae Herba and Bupleuri Radix in cultivation were mainly domesticated from wild species, and various local germplasm were gradually generated by self-breeding. In the long-term of the cultivation, however, the lack of variety selection and unscientific introduction resulted in the existing problems of the intermixtured and degenerated breeds, which have influenced both the quality and yield of the herbs. Besides, there also exist local habitual herbs and adulterants in some areas affecting the drug safety and clinical efficacy. In recent years, with the rise of artificial breeding of medicinal plants, there have appeared a number of elite germplasms with fine traits, thus ensuring the yield and quality of the herbs to a great extent. As the circulatory germplasms are increasing in the market, to explore accurate, effective and quick germplasm identification methods has become an urgent task, since the traditional identification methods can no more meet the demands of germplasm identification, thus to guarantee the interests of farmers, breeding scientists and pharmaceutical companies and to stabilize the medicine market orders. This research tries to explore the molecular and chemical methods for the Schizonepetae Herba and Bupleuri Radix germplasm identification with the purpose of effectively improving the Schizonepetae Herba and Bupleuri Radix germplasm breeding, utilization and development.In this study, by using molecular and chemical methods, the Schizonepetae Herba varieties (Schizonepeta tenuifolia (Benth.) Briq.) of "ZhongJing" series and Bupleuri Radix varieties(Bupleurum chinense DC.) of "ZhongChai" series bred by Wei Jianhe research team, and the main germplasms of Schizonepetae Herba and Bupleuri Radix collected in the current markets have been identified. Identification systems of Schizonepetae Herba and Bupleuri Radix germplasms have been established. The main findings are as follows:1. The Schizonepetae Herba identification system is established by using ITS2 barcoding technology, which can identify Schizonepetae Herba and the adulterants stably and accurately. Taking 12 germplasms of Schizonepetae Herba as experimental samples, ITS2 sequences were extracted following Molecular Identification of DNA Barcoding Standard Operating Procedures. The results showed that the ITS2 sequence length of Schizonepetae Herba was 232bp, the ITS2 sequences from different sources were same, and the ITS2 sequence with 8 adulterants species showed obvious differences which are much larger than the intraspecific differences. The phylogenetic analysis on the Neighbour-Joining tree constructed by Schizonepetae Herba and adulterants of the congener Labiatae plants indicates that the branches between Schizonepetae Herba and the related species and the adulterants are evidently different.2. The best SRAP-PCR reaction system is established providing a foundation for further research on molecular of Schizonepetae Herba. The SRAP-PCR reaction system was optimized using the single factor test and the orthogonal design test and finally the optimum SRAP-PCR reaction system obtained by comparison was in total volume of 25 μL, containing 2.00 mmol/L Mg2+,0.20 mmol/L dNTPs,1.5 U Taq polymerase,0.50 (μmol/L primers,20 ng DNA template and 2.5μL 10×Ex Taq Buffer. The results showed that in descending order the effects caused by the factors on the amplified reaction results were:Mg2+, DNA template, Taq polymerase, dNTPs and primers. By using this system,14 primer pairs which can amplify clear bands were screened from 264 primer pairs. These findings are useful for applying SRAP markers to the study of variety identification of Schizonepetae Herba.3. The SRAP molecular marker identification system of Schizonepetae Herba is established which can identify the new varieties and the main varieties, initially explored the genetic relationship of different sources of Schizonepetae Herba. In this study, sequence-related amplified polymorphism markers (SRAP) were used to identify the 132 samples of 12 germplasms of Schizonepetae Herba. A total of 261 bands were amplified from 14 primer combinations and 89.66% of them (234) were polymorphic. The range of polymorphic percentage of 12 germplasms are from 29.12% to 83.14%, indicating that the degree of internal differentiation of different varieties are significant. The new breeding germplasms have higher uniformity than that of locals. The results suggest that four germplasms (ZJ1#, ZJ2#, AGZZ, YTZZ) can be distinguished by 4 primer combinations (Me1/Em13, Me1/Em17, Me3/Em18, Me7/Em13). Cluster analysis and principal coordinates analysis show that the similarity of different Schizonepetae Herba germplasms has a relation with genetic similarity and but has no relation with geographic positions. The result can be used as reference to identification, production, utilization and genetic diversity research of Schizonepetae Herba.4. The GC fingerprints of Schizonepetae Herba from different sources are established, which provide a scientific basis for variety identification and quality control. The methods of extracting volatile oil are determined through the orthogonal design test with the solid-liquid ratio of 10:1, soaking time of 2 h, extraction time of 3 h. The samples were determined by GC-FID and their fingerprints were set up by Similarity Evaluation System for Chromatographic Fingerprint of TCM (Ver.2004) and analyzed by similarity evaluation, cluster analysis and principal component analysis.10 common chromatographic peaks were obtained from 40 samples which can be well grouped into 4 categories by means of the cluster analysis and PC A.5. The SSR molecular marker identification system of Bupleuri Radix cultivated germplasms is established which can identify new varieties and selected strains. A total of 9 primer pairs with high polymorphism and clear bands were selected from 50 primer pairs. The two selected strains and four other Bupleuri Radix cultivated germplasms were amplified. According to the specific SSR bands, different germplasm can be distinguished, a dendrogram on the basis of genetic distance was constructed, and thus, all of the samples were classified into 4 groups:B. scorzonerifolium cultivated in Heilongjiang and Chuanhongchai No.1 strain as one clade; Chuanbeichai No.l strain and Zhongchai No.l cultivar clustered as another category; cultivated germplasms from Sichuan Fengshun and Rongxian clustered separately as individual classes. This study helps to identify Bupleuri Radix germplasms and provide a reference for future breeding.Above all, this study, taking as experimental materials the newly-bred varieties of the Schizonepetae Herba and the Bupleuri Radix and main cultivated germplasms of Schizonepetae Herba and Bupleuri Radix collected in the current market, established the identification systems of Schizonepetae Herba and Bupleuri Radix by using molecular and chemical methods. The findings can provide the technical support and scientific basis for germplasm protection, variety breeding and quality control of the two herbs. |
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