Fitohemijska i molekularno-genetička karakterizacija populacija kičice (Centaurium erythraea Rafn) sa Balkanskog poluostrva
Phytochemical and molecular characterization of centaury (Centaurium erythraea Rafn) populations from the Balkan Peninsula
Doctoral thesis (Published version)
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Centaury (Centaurium erythraea Rafn) is a medicinal and pharmacologically attractive plant species which has a wide range of biological activity. The main objective of presented study was to estimate genetic and phytochemical diversity of centaury within the Balkan Peninsula. With that purpose, seed collection of natural centaury populations was established. The study included 43 populations of C. erythraea, among which 41 originated from the Balkan Peninsula. Molecular marker techniques, such as RAPD (Random Amplification Polymorphic DNA) and TRAP (Target Region Amplification Polymorphism) were optimized to investigate the genetic diversity among these populations. DNA used in these analyses originated from in vitro grown plants to ensure the authenticity of centaury DNA by eliminating the possibility of its contamination by extraneous DNA. To acquire sufficient informational characters, 16 decameric primers were applied in RAPD assays, which produced 408 different PCR products, while TRAP assays included the combinations of 3 specific and 3 arbitrary primers, resulting in 767 fragments of DNA. Specific primers used for TRAP analyses were constructed according to the RNA sequences, showing high homology with DNA sequences published in electronic data bases for following enzymes: geraniol 10-hydroxylase, cytochrome P450 reductase and secologanin synthase. Molecular markers provided a high percentage of polymorphism. The informativeness of single primers (RAPD) of primer combinations (TRAP) was confirmed by various parameters. Low within-population genetic variability of natural populations of centaury could be explained by the high levels of selfing or geitonogamy in the process of fertilization and by strong genetic drift. Due to the fact that centaury grows in small, fragmented populations and that gene flow between them probably does not exist, a high genetic differentiation of analyzed populations was expected and this was confirmed by molecular markers. Moreover, molecular markers Резиме/Summary efficiently differentiated diploid populations from tetraploids. Tetraploid populations were clearly clustered into groups related to their geographic origin, especially when RAPD markers were analyzed. Genetic similarity data between the Balkan Peninsula populations, obtained with either RAPD or TRAP markers, significantly correlated with the geographic distances between localities of their origin. Identification and quantification of secondary metabolite compounds, secoiridoid glycosides, highly contributed to the estimation of diversity of centaury populations in the analyzed region. A dominant secoiridoid glycoside in aerial parts of plants grown under greenhouse conditions was swertiamarin, which pointed to the interpopulation variability of centaury. Furthermore, a significant correlation between the secoiridoid glycosides content and genetic similarity of populations has been obtained, though a higher correlation was recorded for TRAP markers. The genetic and phytochemical diversity estimation of centaury from the Balkan Peninsula provides a basis for future biodiversity conservation efforts and also for high-productive genotype selection and field production improvement.
Keywords:Centaurium erythraea; Balkan Peninsula; Habitat fragmentation; Populations; Molecular markers; RAPD; TRAP; Genetic variability; Secoiridoid glycosides
Source:University of Belgrade, Faculty of Biology, 2012, 1-149
- Physiological, chemical and molecular analysis of the diversity of selected rare and endangered plant species and application of biotechnology for ex situ conservation and production of biologically active compounds (RS-173024)