| As one of the largest family in angiosperm and an important cultivation plant in horticulture and flower, Orchid has been cultivated in China for about 2000 years. Orchid resources are quite rich, totally, there are nearly 17,500 species of about 800 genera in the world and 1247 species of 173 genera in China. Because of its high value in ornamental, medical and economic aspects, studies such as investigation of germplasm resources, introduction and domestication of wild species and analysis of genetic diversity have been done on Chinese Orchids. However, because of the speciality of orchid species, researches on Chinese orchids have been greatly limited, which is causing the continuous losses of some rare and wild species. Presently, it is extremely urgent to investigate the germplasm resources of Chinese orchids, and to understand their characteristics in morphology, biology and genetics by using methods such as tissue culture, biochemistry, cellular and molecular biology. Here, totally 21 strains of 7 species of cymbidium subgen.Jensoa from Guizhou province, China were analyzed by using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and PCR based restriction fragment length polymorphism (PCR-RFLP) analysis of Chloraplast and Mitochondrial DNA as well. Results are as the followings:1. Twenty one subgen.Jensoav strains of Chinese orchid from Guizhou province were analyzed by RAPD and results showed that 28 out of the 40 10bp-length random primers could reveal the polymorphism among the selected strains. Totally 142 bands were obtained, among those bands,128 are polymorphic ones (90.14%). One RAPD primer BO319, which could get clear bands and had good attribute in polymorphism analysis was selected and A, T, C or G was added to its 3'end, and thus four new primers were generated. Ten extended-RAPD (ERAPD) primer pairs were then randomly matched from the 4 new primers. Eight out of the ten ERAPD primer pairs could amplify more specific bands than RAPD primer did, while amplification efficiency of the other two pairs were decreased dramatically. Fifteen out of the all 26 bands amplified from the 21 samples with the 8 ERAPD primer pairs showed polymorphism. One band, with a size of 2.5 kb was obtained from all the samples, it might associate with genes which control a certain critical feature of Orchids. Genetic similarities of the 21 strains were calculated from all the 168 bands for further cluster analysis. Results showed that all the samples were divided into 3 groups (namely, group A, B and C) by the two molecular markers at a level of genetic similarity 0.537. In addition, results also indicated that longibracteatum and Lianpan had a close phylogenetic relationship in goeringii group, while sinense was similar to kanran in ensifolium group.2. Eight out of the 18 pairs of ISSR primers could produce differential bands among the 21 subgen.Jensoav strains of Chinese orchids from Guizhou province and 42 out of the 78 amplified bands were polymorphic ones. Genetic similarities were from 0.613 to 0.862 among the samples and the highest one was from two strains of Chunlan species. Cluster analysis indicated that all the 21 strains were divided into two groups, namely, goeringii and ensifolium. Moreover, strains in the goeringii group were again divided into subgroups Al and A2 at higher genetic similarities level. The fact that Lianpan shared a highest genetic similarity with goeringii revealed that the two strains had the closest phylogenetic relationship.3. Genomic DNAs from all the samples were amplified with cytoplasm universal primers of 6 chloroplast and 4 mitochondrial. Results showed that 4 chloroplast and 2 mitochondrial primers could generate bands with good quality. Amplified products were digested with 8 restriction endonucleases (Hinfâ… , Rsaâ… , EcoRâ… , EcoRâ…¡, BamHâ… Hpaâ… , Pstâ… and Xhoâ… ). Twelve of the PCR products produced by the 4 chloroplast universal primers were digested into 47 bands, and 39 bands showed polymorphism. Seven of the PCR products amplified by the 2 mitochondrial universal primers were digested into 27 bands, and around 85.19% of the bands were polymorphic ones. Genetic similarities of the tested samples were calculated from all the obtained 74 bands and cluster analysis was carried out. Results showed that the genetic similarities were from 0.625 to 0.925 and the 21 subgen.Jensoav strains of Chinese orchids were thus clustered into group A, B and C. Furthermore, the fact that goeringii, longibracteatum and Lianpan shared a highest genetic similarity indicated that they had a closer genetic relationship.4. As a whole, this study indicated that RAPD (ERAPD), ISSR and PCR-RFLP are useful tools for the characterization of germplasm resources of orchids. The study can also offer theoretical references for the effective usage of orchid resources, popularization of superior strains, protection of intellectual properties and establishment of industrial policies as well.
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