Rapid Propagation Technique And Isolation Of Microsatellite Markers For Bletilla Striata

The genus Bletilla, composting about six species, is endemic to Asia with a distribution pattern from N Myanmar and Indochina through China to Japan. Bletilla not only has high ornamental value, but also is one of our traditional Chinese medicines. Bletilla striata (Thunb.) Reichb.f. is the most widely used species among the genus. At present, the cultivation of B.striata is only limited to the temperature areas of North America and Europe for the horticultural purpose, and the using of pseudobulbs of B. striata for other purpose especially for medicinal purpose is basically based on wild resources. Undoubtedly, the wild resource of B.striata will rapidly decrease in Eastern Asian Countries due to over collections as well as habitat destruction and fragmentation. Now research found that B.ochracea Schltr. alm ost has the same drug efficacy with B.striata. So, it can take the place of the Bletilla striata, which can ease the pressure of the B.striata demand. Microsatellite marker, with many advantages, has been applied extensively in the field of genetic diversity analysis, genetic map construction, population genetics investigation and so on. The aim of this study not only find out the best proceeding of culturing, but also developed microsatellite primers to investigate its genetic diversity for making concrete protection strategy. This paper contains three parts:1. Seedlings growth and asymbiotic germination of B. ochracea seedsOrthogonal test design L9(34) was used to study the rapid propagation techniques of mature seeds of B.ochracea. The results showed that the potato, sucrose, NAA and6-BA have not significant impact on the sprouting rate of seed indicating that the requirements of mature seeds on medium low, easy germination; but the sugar and potato have a significant impact on the development of seedlings. The optimal conditions for seedling development is that the medium added with sucrose10g/L+potatoes100g/L+NAA0+6-BA0.5g/L.2. Seed embryo growth courses of three orchid species Microexamination were carried out on the embryo development of the seed of B. ochracea、 Dendrobium fimbriatum、Pleione bulbocodioides, and similarities and differences were studied. During the process of seed germination, seed embryo of B. ochracea broke through the midst of the seed coat or come out from one end. We have found there were two routes for the embryo growths of P. bulbocodioides and D. fimbriatum. One of the route was the embryo become green after breaking through the seed coal, the other was the embryo turned green before breaking through the seed coat at one end away from residues suspensor after turning green, and then form protocorm. The globules grew into seedlings through their protocorms.3. Isolation of microsatellite markers for Bletilla striata and cross-amplification in other related species.In this study, the microsatellite primers were obtained by using the method of enrichment by magnetic beads. Designed56pairs of microsatellites sequence by PrimerPremiers5.0and SSR Hunterl.3and thirteen primer pairs have specific and right loci. Using the fluorescently labeled primer for amplification, nine primer pairs showed clear bands and expected size. These9primer set were characterized in two wild populations of Bletilla striata and one wild population of Bletilla ochracea. The number of alleles per locus ranged from1to12. The expected and observed heterozygosities ranging from0to0.7646and0to0.950in Bletilla striata respectively. In Bletilla ochracea, the expected and observed heterozygosities ranged from0.296to0.871and0.05to1, respectively. These nine polymorphic SSR markers provide useful method for studying population genetics and evolutionary history of the genus Bletilla, which would provide guarantee for making valid protection strategy. The high interspecific amplification of these markers suggests their potential value to study other congeneric species of orchids.