| Salvia miltiorrhiza Bge is a traditional Chinese herb plant, its root has been used for centuries to treat diseases such as angina pectoris, cerebral vascular diseases, degenerative diseases, acute or chronic liver injuries, and has extensive and important curative effects in practice. While quantity need for this medicinal material increased and wild resource dropped off year after year, the planting area was added, but there existed severity farrago in cultivate regions. Now the plant region mainly distributed in Sichuan, Shanxi, Shangdong, Henan and Hebei province in China. Research about genetic diversity and genetic structure of different S. miltiorrhiza geographical populations in China would benefit for understanding of the genentic base and genetic relations among geographical populations, and also benefit to the germplasm collection, sonservation, utilization, and improvement of S. miltiorrhiza.In this study, S. miltiorrhiza germplasms from nine geographical populations in China were analysised by morphological, cytology, molecule biology method. Morphological variation, the floral biology and breeding system of S. miltiorrhiza was preliminary studied, the genetic diversity and relation between populations was investigated Moreover the suitable variety for cultivation in the Shandong region was selected based on root character and the content of active ingredients. The results are as follows:.1. There were extensive variations in morphology characters indifferent geographical populations of S. miltiorrhiza. Among forty-two morphologic traits, the most significant variance lay in petiole color and length, leaf width and length, leaf shape index, flower color, plant highth and plant breadth, ramification number. Inter-/intro-population variation occurred significantly. Genetic differentiation between and within five eco-geographical populations of S. miltiorrhiza was estimated by Shannon-Wiber diversity index, which showed that genetic variance existed within and among populations with same frequency. Comparing with Shannon-wiber diversity index for nine populaitions in China, the genentic diversity of Tai'an was the highest, Heze and Zhongjiang populution were less. Fourty-two morphological traits could be divided into seven groups Principal component analysis(PCA) showed that the forty-two characters can be consolidated for the seven principal components and their accumulative contribution ratio amounted to 70.17%. according to the relativity between characters and principal component analysis , sixteen major discriminating charactics were selected, synthesizing flowr factors, blanches and leaves factors. These characters would better used to analysis and cluster for S. miltiorrhiza replacing 42 characters. Based on UPGMA analysis of Euclidean distance among individual cultivars of S .miltiorrhiza, germplasms were clustered into 6 groups. Moreover based on the characteristics of each group and the resultof PCA the classification of S. miltiorrhiza was taked out which would benenfit for assession of S.miltiorrhiza germplasm in future.2. Mitosis study showed that the chromosome numbers of nine geographical populations of S. miltiorrhiza were also 2n=16, no polyploidy cells and B chromosome were observed. The karyotype of Zhongjiang population was 2n = 16 = 6m + 10sm, and the karyotype of other population were 2n = 16 = 8m + 8sm. Among six populations, two chromosomes with satellite was observed. Nine population could be cluster into three type based on the relative chromosome length and arm ratio. Meiosis study showed that all the sixteen could be paired into eight bivalents at metaphase I, and high frequency of cytomixis were observed in prophase I.3. Fifty-six S. miltiorrhiza lines from five different geographical populations located at Tai'an, Linyi, Laiwu, Heze and Weifang in Shandong province of China were studied by ISSR and SRAP markers. The results indicated that percentage of polymorphic loci (P) on species level was 97.72%, 91.46% respectively, it indicated that there were abundant genetic diversity on species level. Average 16.6 polymorphic bands with in every ISSR primer in each population were detected and the percentage of polymorphic loci (P) was 60.63%. Average 9.36 polymorphic bands in every SRAP primer in each population were detected, the percentage of polymorphic loci (P) was 57.07%. Genetic differentiation between and within five eco-geographical populations of S. miltiorrhiza was estimated by genetic differentiation coefficient (Gst=0.1352, 0.1892) which showed that 13.05%, 20.19%genetic variance was among populations revealed by ISSR and SRAP markers. More than 80% genetic variance existed within populations. The genentic diversity Comparing with percentage of polymorphic loci, Nei's gene diversity index and Shannon's information index, the genentic diversity from Tai'an was the highest, Linyi and Laiwu was higher and secondly Heze with the last. UPGMA cluster analysis indicated that Laiwu population and Linyi population were higher in genetic identity which was nearer in genetic relationships, the genetic distance between heze and above two populations was also near therefore the three populations could be cluster into one group, however the genetic distance between Tai'an population and other four populations were farther therefore into another group. Furthmore the genetic relationships beween Laiwu population and other populations was different revealed by ISSR and SRAP markers.. Based on Nei's unbiased measures of genetic identity, UPGMA cluster analysis measures of five eco-geographical populations. It indicated that there was a correlation between genetic relationships and geographical distance among the populations. Based on the study of population genetic structure, owing to the highest genetic diversity, Tai'an population should be given a high priority consideration in Salvia miltiorrhiza populations in situ germplasm conservation strategies.Moreover4. Sixty-two S. miltiorrhiza from five different geographical populations located different province of China were studied by ISSR and SRAP markers. The results indicated that a total of 120 and 122 loci were respectively amplified from S. miltiorrhiza with five ISSR primers and six SRAP primers, the percentage of polymorphic loci (P) on species level was 100%, 90.10% respectively by two different markers which revealed that there were abundant genetic diversity on species level. Average 14.48 and 11.27 polymorphic bands with in every ISSR and every SRAP primer in each population were detected with the average percentage of polymorphic loci (P) was 60.63%, 57.07% respectively. Genetic differentiation between and within five eco-geographical populations of S. miltiorrhiza was estimated based on genetic differentiation coefficient (Gst), which showed that 80% genetic variance existed within populations, and less than 20% genetic variance was among populations revealed by ISSR and SRAP markers. Comparing with percentage of polymorphic loci, Nei's gene diversity index and Shannon's information index, the genentic diversity of Lushi population was the highest, secondly Tai'an population, Shangruo population from Shanxi province and Neixiang population from Henan province were high too with the last.Zhongjiang population from Sichuan province. UPGMA cluster analysis indicated that Zhongjiang population and Shangruo population also higher in genetic identity were clustered into one group, the genetic distance between two populations of Henan province was nearer , Tai'an population were relative independent populations, but Tai'an population and two populations of Henan provinc relative near therefore the three populations could be cluster into another group. Based on Nei's unbiased measures of genetic identity, UPGMA cluster analysis measures of five eco-geographical populations. indicated that there was a correlation between genetic relationships and geographical distance among the populations.5. Different lines of Salvia miltiorrhiza were separated in field based on the biological characteristics, the overground biological characters and root character and contents of active ingredients including Tanshinoneâ…¡A, Cryptanshinone and Salvianolieaeid B of them were evaluated. The results showed that unifoliate round leaf typr of S. miltiorrhiza have better agronomic characters , weighter root and higher active ingredients, it was more suitable for cultivation in the Shandong region.6. Floral biology of Salvia miltiorrhiza showed that the flowers of S. miltiorrhiza were bisexual and never-ending, the blossom of a single flower could last approximately 5?7 days. Pollination peak last from 2 days before blossom to 6 hours after blossom, and the higher stigma receptive stage was the same time. Self-pollination was possible by temporal and spatial superposition of male and female organs for 2 day within the same flower. Seed set percentage of autogamous selfing in isolation without emasculation condition and geitonogamous selfing mainly pollinated through different flower in one plant. The results indicated that no apomixis occurred, the breeding system was outcrossing with self-compatibility and pollinators were needed based on the pollen-ovule ratio(p/o), out-crossing index and the results of emasculation, bagging and artificial pollination studies. The most frequent floral visitors were bees. It was inferred elementary that S. miltiorrhiza belong to often cross-pollinated plant. The crossing method and breeding strategy were discussed. |
