| The genus Salvia L., the largest member of the family Lamiaceae, includes approximately1050species distributed in tropical and temperate areas of both old and new world. There are78species,24variations,8forms in China, distributing in most areas especially the southwest regions. Among the large number of Salvia in China,36accessions (including variations and forms) have the medical value. As one of the famous medicinal materials, Salvia miltiorrhiza Bunge. has been recorded by "Pharmacopoeia of the Peoples Republic of China". With the rapid increasing of the demands in S. miltiorrhiza, different sources were sold as S. miltiorrhiza in the herbs markets, and which was affected seriously its clinical application and development. Therefore, it is extremely important to establish a scientific, reliable, quick and easy identification system for identification. In recent years, the quality of S. miltiorrhiza degraded severely. However, it is impossible to have a good variety without excellent germplasm, and the understanding of the genetic basis and genetic relationship among different species is the premise for exploiting these germplasm resources effectively. In this study, we studied the identification and relationship from superficial, micro, chromosome morphology and molecular biology by using S. miltiorrhiza and its wild relative resources as materials. The intraspecific and interspecific genetic diversity were analyzed to reveal the degree of genetic basis, genetic differences and genetic distance in different populations. The main aim of this research is to provide proofs for the identification and utilization of S. miltiorrhiza germplasm resources and lay a good foundation for further in-depth research and genetic improvement. The main results are shown as follows:(1) Based on the investigation and collection of wild resources combined with specimen data,18morphological traits of the31taxa on the genus Salvia in Sichuan province were observed and measured. The genetic diversity was analyzed according to the apparent morphology. The results of the quantitative traits showed the varieties were significant in leaves and the most were petiole length/leaf length. The followed were petiole length, leaf width and leaf length. And the corolla length/calyx length was the least. About the quality characters, multiple types were appeared in each biological traits. Leaf length, fertility of anther chamber, petiole length/leaf length and number of years were the characteristic trait for Salvia based on the PCA analysis. And those traits were conducted by R cluster and Q cluster analysis. The result of R cluster analysis shows that18morphological traits are independent and could be taken as the standard for classification; the results of Q cluster analysis showed that the31taxa were gathered into4groups which are consistent with the traditional taxonomy on the genus. However, the system location of S. kiaoemetiemsis f. pubescens needs more new data to define. According to the results, the genetic relationships among these taxa are analyzed which can give evidences for system classification, identification of genetic relationships and utilization of germplasm resources on Salvia in Sichuan.(2) The cross sections of14materials, containing S. miltiorrhiza from seven provinces, S. brevilabra, S. omeiana, S. cavaleriei var. simplicifolia, S. tricuspis, S. trijuga, S. yunnanensis and S. przewalskii were obtained by using paraffin sectioning and the optical microscope was used to characterize the structure. The results showed that there was much commonness in the materials studied on the microscopic structure of roots, such as wide secondary cortex, narrow phloem, well-developed xylem, obvious cambium and so on. But the differences exist in rhytidome, cork layer thickness and suberification degree, number and size of the canal in xylem and also the tactic mode. These differences and the build search of transverse section features on the roots could be as the anatomical evidences for the identification of the S. miltiorrhiza from the mixed medicinal markets, and also may be considered to be the main features for the classification of Salvia.(3) Observations were made on chromosome morphology and behavior during meiosis of PMCs (pollen mother cells) for different origins of S. miltiorrhiza and its related species in the genus Salvia. Results show that the basic chromosome number is x=8and there were diploid and tetraploid in the genus Salvia. According to the chromosomes pairing in metaphase I, except that S. przewalskii and S. brevilabra are tetraploids, the others are diplonts. The chromosome configurations were different in each sample, and the rod bivalents occupied a relatively high proportion compared with the ring bivalents. With the highest proportion of ring bivalents, the switching frequency and intraspecific variation were the highest in S. flava, and the lowest was S. miltiorrhiza from Shanxi. There was a B chromosome in S. brevilabra and S. miltiorrhiza from Henan respectively. Additionally, the abnormal behaviors of the chromosomes in meiosis observed may have effects on the development of pollen grains in S. miltiorrhiza from Henan.(4) Among70random primers examined,27randomly amplified polymorphic DNA (RAPD) primers produced248extending and repeatable bands, of which207bands (83.47%) were polymorphic and the average number of Polymorphic bands was7.67. All of the data revealed abundant genetic diversity in the genus of Salvia. According to the genetic similarity coefficient obtained from RAPD fingerprinting for each primer, we can distinguish these samples.18taxa were clustered into four groups based on RAPD markers and all S. miltiorrhiza and S. miltiorrhiza f. alba were in a cluster. The results showed that there were slighter genetic diversity and narrower genetic backgrounds within intra-species, and we can easily distinguish S. miltiorrhiza obviously from the plenty of Salvia species by RAPD. The clustering results based on RAPD were inconsistent with traditional morphological classification results, and the main differences were in the system status of S. omeiana and S. tricuspis.(5) An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL-F, and psbA-trnH from the chloroplast genome, and ITS, ITS1, and ITS2from the nuclear genome) and three combinations (rbcL+matK, psbA-trnH+ITS1, and trnL-F+ITS1). However, the result did not show ideal target as COX I in animals. The discriminatory capabilities of these regions were evaluated in terms of identification efficiency via a tree-based method. ITS1was superior to the other marker for discriminating between species, with an accuracy of81.48%, and followed the trnL-F with an accuracy of77.77%. Among the three combinations, the highest was trnL-F+ITS1with an accuracy of74.74%and the three combinations did not increase species discrimination. In addition, in the examined fragments, ITS1was the only one sequence which can distinguish all S. miltiorrhiza samples. Finally, we recommend ITS1as the DNA barcoding for identifying Salvia species, especially S. miltiorrhiza. |
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