Specific Molecular Markers For The Inter-species And Intra-species Genetic Analysis On Calycanthus Chinensis And Machilus Thunbergii

The purpose of the dissertation is to explore appropriate molecular markers in plant genetic diversity area, so that to provide convenient, practical and dependable molecular methods for plants protection and for the authentication of species and cultivars.The study includes two parts. In the first part species-specific molecular primers were developed for the authentication of an ornamental landscape species, Calycanthus chinensis Cheng et S. Y. Chang, which is classified as second-grade protected wild plants in the Chinese Plant Red Book; In the second part ISSR (Inter-Simple Sequence Repeat) marker assays and trn L-F sequencing technique were used on four cloning types of Machilus thunbergii Sieb.et Zucc for cultivaridentification and genetic variation study.1) Calycanthus chinensis, an endangered species endemic to China, is cultivated as an ornamental landscape tree in China. In this study, ISSR (Inter-simple sequence repeats) were applied to detect C. chinensis from its closely related species. A unique 748-bp band was found in all accessions of C. chinensis. SCAR (Sequence characterized amplified regions) markers were created by cloning and sequencing the specific band, and designing a pair of primers to amplify the band of 748 bp. Diagnostic PCRs were performed using the primer pair with the total DNAs of C. chinensis, Chimonanthus species and C. floridus as templates, with only C. chinensis being able to be amplified. This amplification is not only rapid (results can be obtained in less than 3h), but is also easy to perform. Hence it is an easy, feasible and dependable method for identifying C. chinensis.2) Based on introduction and selective breeding of Machilus thunbergii, genetic variation of four cloning types of M. thunbergii were determined using ISSR marker assays. Seven out of 28 ISSR primers could generate reproducible polymorphic fragments. The ISSR analysis revealed a total of 83 DNA bands, of which 42 were polymorphic (the percentage of polymorphic bands, PPB = 50.5%). The average DNA bands amplified by each primer were 11.86. A dendrogram was...