Abstract
Random amplified polymorphic DNA (RAPD) analysis is a simple and reliable method used to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of the assay. In this study, we analyzed the effects of different concentrations of primer, magnesium chloride, template DNA and Taq DNA polymerase to develop and standardize a RAPD protocol for Centaurium species. The optimized PCR reaction mixture included: 50 ng of DNA extracted using a CTbased protocol, 2.5 mM MgCl2, 7.5 pmol primer and 2 U of Taq polymerase in a final volume of 25 ?l. Each of the five primers used in experiments (OPB11, OPB15, OPB18, OPF05 and OPH02) generated reproducible and distinguishable fingerprinting patterns of four Centaurium species. The obtained optimized RAPD protocol and the selected primers are useful for our further work in the genetic diversity studies of Centaurium species.
Highlights
The majority of Centaurium species, are a rich source of bitter secoiridoid glycosides, xanthones and β-monoglycosides which are reported to have a wide range of bioactivity, with antifungal and antibacterial activity being the most investigated ones
The obtained in vitro plant material is free of viruses and other pathogens, the contaminations of the isolated plant DNA with foreign DNA is excluded
This is especially important when isolated DNA is further used as a template for Random amplified polymorphic DNA (RAPD) analysis, where small unspecific primers are used
Summary
The similar phenotypic characteristics of different Centaurium species perplexed botanists and herbalists during the last century (Pringle, 1987). Some species of this genus are often found alongside each other in the same habitat where interspecies hybrids arise readily, causing morphological variability and resulting in taxonomic divergences (European Medicines Agency, 2009; Mansion, 2004; Mansion et al, 2005). In areas of the British Isles and northern Europe, hybridization between Centaurium erythraea Rafn subsp. Littorale has resulted in the production of a new allohexaploid species Centaurium intermedium (Wheldon) Druce (Ubsdell, 1979) Turner) Gilmour subsp. littorale has resulted in the production of a new allohexaploid species Centaurium intermedium (Wheldon) Druce (Ubsdell, 1979)
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