Studies on Chloroplast Dna Restriction Fragment Length Polymorphisms of Three Species of Guizotia Casso (G. Abyssinica,G.Scabra Ssp. Scabra and G.Scabra Ssp. Schimperi)

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Addis Ababa University


Chloroplast DNA from three Guizotia species (G. abyssinica, G. scabra ssp. scabra and G. scabra ssp. schimperi) were isolated using sucrose gradient centrifugation method and Dynabeads DNA DIRECTTH System I. Each of the DNA extracted by sucrose gradient method was separetely digested with BamHI, EcoRI and HindIII restriction endonuclease enzymes, recognizing 6 base for 4hr. The digestion products were then loaded on 0.8% agarose gel and electrophoresed at 30-50 volts for 3-4 hours. After visualizing with o. 5l1g/ml of ethidium bromide solution and photographed using polaroid films, the fragment bands were compared. In addition, the DNAs extracted by Dynabeads DNA DIRECTT1 -{ System I method were used for PCR amplification of chloroplast DNA non-coding region (trnL (UAA) intron) using Primer C and D of Taberlet et al. (1991). The amplified chloroplast DNA non-coding "region, I-las digested with Sau3AI restriction endonuclease enzyme recognizing 4 base pairs at 37°C for 2 hr. and electrophoresed in 2% agarose gel at 100 volts for 2 hr. After being visualized with 0.5I1g/ml ethidium bromide solution and photographed using polaroid films, the fragment bands I~ere compared. In both cases, detectable variation I~as not observed among the Guizotia species compared, indicating that these species are either closely related and recently diversified or their chloroplast DNAs are evolving at a relatively slO\~er rates in comparison to the rates of cpDNA evolution in other angiosperm species.